TW201418472A

TW201418472A - Marker consisting of plasma microRNA and a new method for diagnosis of hepatocellular carcinoma

Info

Publication number: TW201418472A
Application number: TW101142093A
Authority: TW
Inventors: Jia Fan; Jian Zhou; Zhi Dai; Lei Yu; Jie Hu; Zheng Wang
Original assignee: Zhongshan Hospital Fudan Univ
Priority date: 2012-11-12
Filing date: 2012-11-12
Publication date: 2014-05-16

Abstract

The present invention relates to a kit for diagnosing hepatocellular carcinoma consisting of plasma microRNA, a kit containing the same, and a new method therefor. The marker for diagnosing hepatocellular carcinoma consists of a plurality of nucleic acid molecules, each nucleic acid molecule encoding at least one microRNA sequence, preferably consists of nucleic acid molecules encoding hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR-27a, hsa-miR-801 and hsa-miR-1228. The kit can be used for diagnosing hepatocellular carcinoma, especially early hepatocellular carcinoma, and also for discriminating plasma of at least one hepatocellular carcinoma patient from that of at east one healthy individual, at east one chronic hepatitis B patient, or at east one cirrhosis patient.

Description

由血漿mircoRNA組合成之肝癌診斷標誌物及診斷肝癌之新方法 A diagnostic marker for liver cancer composed of plasma mircoRNA and a new method for diagnosing liver cancer

本發明係關於一種一種由血漿microRNA組合成的肝癌診斷標誌物及一種診斷肝癌(特別是早期肝癌)的新方法，所述的血漿microRNA包含hsa-miR-122，hsa-miR-192，hsa-miR-21，hsa-miR-223，hsa-miR-26a，hsa-miR-27a以及hsa-miR-801。 The present invention relates to a liver cancer diagnostic marker composed of plasma microRNA and a novel method for diagnosing liver cancer (especially early liver cancer), wherein the plasma microRNA comprises hsa-miR-122, hsa-miR-192, hsa- miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR-27a and hsa-miR-801.

肝細胞癌(Hepatocellular carcinoma，HCC)是世界範圍內最常見的實體惡性腫瘤之一。其代表了肝癌的主要組織學類型，可能占所有肝癌病例的70%~85%。世界每年會出現大約600000個新病例，和幾乎相同數目的死亡病例，這反應出對這種疾病缺乏有效的早期診斷和治療(Thorgeirsson,S.S.and Grisham,J.W.(2002)Nat Genet 31,339-346；Jemal A et al.(2011)CA Cancer J Clin 61：69-90.；Bosch F.X.et al(2004)Gastroenterology 127,S5-S16；Perz J.F.et al.(2006)J Hepatol 45,529-538)。 Hepatocellular carcinoma (HCC) is one of the most common solid malignancies worldwide. It represents the main histological type of liver cancer and may account for 70% to 85% of all liver cancer cases. There are approximately 600,000 new cases and almost the same number of deaths each year in the world, reflecting the lack of effective early diagnosis and treatment of this disease (Thorgeirsson, SS and Grisham, JW (2002) Nat Genet 31, 339-346; Jemal A et al. (2011) CA Cancer J Clin 61: 69-90.; Bosch FX et al (2004) Gastroenterology 127, S5-S16; Perz JF et al. (2006) J Hepatol 45, 529-538).

HCC是一類預後很差的癌症。此種疾病患者的預後取決於疾病分期。HCC患者未經手術治療，5年生存率為<5%，手術後生存率為60%~70%。腫瘤尺寸<2 cm且採用外科手術切除的患者的5年生存率能夠達到86%。但是，沒有經過任何治療的早期癌症患者(腫瘤尺寸<5 cm)的3年生存率僅為17~21%。這證明了早期癌症檢測對於治療以及提高患者生存率都是非常重要的。(Tang,Z.Y.(2001)World J Gastroenterol 7,445-454；Chambers,A.F.et al.(2002)Nat Rev Cancer 2,563-572；Motola-Kuba D.et al.(2006)Annals of Hepatology 5,16-24)。 HCC is a type of cancer with a poor prognosis. The prognosis of patients with this disease depends on the stage of the disease. Patients with HCC were treated without surgery, with a 5-year survival rate <5% and a postoperative survival rate of 60% to 70%. Patients with tumor sizes <2 cm and surgically resected have a 5-year survival rate of 86%. However, the 3-year survival rate for early cancer patients (tumor size <5 cm) without any treatment is only 17-21%. This proves that early cancer detection is very important for treatment and to improve patient survival. (Tang, Z.Y. (2001) World J Gastroenterol 7, 445-454; Chambers, A. F. et al. (2002) Nat Rev Cancer 2, 563-572; Motola-Kuba D. et al. (2006) Annals Of Hepatology 5,16-24).

雖然近年來早期偵測與手術移除肝細胞腫瘤可顯著地提升患者的存活率，但大多數的腫瘤在癌化過程(即，非致死階段)中仍無法被早期偵測出。在藉由相對正常的肝功能指數以及經過有效的臨床分期系統診斷的受控制的腫瘤病變確認的HCC患者中，只有約10%~20%目前適用於外科手術。此外，經手術切除的患者仍很可能會轉移/復發，且術後5年存活者亦只有30%~40%。 Although early detection and surgical removal of hepatocellular tumors in recent years can significantly improve patient survival, most tumors are still not detected early in the cancerization process (ie, non-lethal stage). Of the HCC patients confirmed by the relatively normal liver function index and controlled tumor lesions diagnosed by an effective clinical staging system, only about 10% to 20% are currently suitable for surgery. In addition, patients who have undergone surgical resection are still likely to have metastasis/relapse, and only 30% to 40% of survivors are 5 years after surgery.

一個明確的肝癌診斷經常以組織學確認為基礎。組織可以通過細針穿刺或生物檢體來取樣。一些肝癌分化良好，這意味著它們幾乎完全由生長的、成熟的肝細胞組成。因此，這些癌症在顯微鏡下看起來非常類似於非肝癌組織。此外，並非所有的病理學家都能夠識別分化良好的肝癌和正常肝組織的細微差別。一些病理學家會把肝癌誤認為肝腺癌。腺癌是一種不同類型的癌症，它從肝外起源。轉移性腺癌和原發性肝癌治療方法不同，因此，需要早期檢測以區分這些不同類型的腫瘤，指導HCC患者的治療，以改善患者長期生存率。 A clear diagnosis of liver cancer is often based on histological confirmation. Tissue can be sampled by fine needle aspiration or biopsy. Some liver cancers differentiate well, which means they are almost entirely composed of growing, mature liver cells. Therefore, these cancers look very similar to non-hepatocarcinoma tissues under the microscope. In addition, not all pathologists are able to identify subtle differences between well-differentiated liver cancer and normal liver tissue. Some pathologists mistake liver cancer for liver adenocarcinoma. Adenocarcinoma is a different type of cancer that originates from the outside of the liver. Different treatments for metastatic adenocarcinoma and primary liver cancer require different tests to distinguish these different types of tumors and guide the treatment of HCC patients to improve long-term survival.

肝穿刺或生物檢體的最常見風險是出血，因為肝癌是含血管非常豐富的腫瘤。在許多情況下，可能沒有必要進行組織生物檢體或穿刺。如果患者具有發生肝癌的危險因素(例如，肝硬化，慢性B型肝炎，或慢性C型肝炎)，血漿中α-胎蛋白(AFP)的水準顯著升高，並且有相符合的影像學診斷，醫生幾乎可以不通過生物檢體就能確定病人患有肝癌。目前，AFP是唯一的用於早期診斷肝癌的血清標誌物(Mizejewski,G.J.(2003)Expert Rev Anticancer Ther 2, 709-735；Paul,S.B.et al.(2007)Oncology 72,Suppl.1,117-123)。然而，這種單一的標誌物靈敏度低，且經常出現假陽性結果，例如在大量的肝硬化患者中，AFP也會升高。此外，血清AFP僅能檢測出60%的肝癌患者。因此，發現早期診斷HCC的新的分子標誌物以及開發靈敏的檢測方法具有重要意義。 The most common risk of liver puncture or biopsy is bleeding, because liver cancer is a tumor that is very rich in blood vessels. In many cases, it may not be necessary to organize a biopsy or puncture. If the patient has a risk factor for developing liver cancer (eg, cirrhosis, chronic hepatitis B, or chronic hepatitis C), the level of alpha-fetoprotein (AFP) in plasma is significantly elevated and there is a consistent imaging diagnosis. It is almost impossible for a doctor to determine that a patient has liver cancer without passing through a biological specimen. Currently, AFP is the only serum marker for early diagnosis of liver cancer (Mizejewski, G.J. (2003) Expert Rev Anticancer Ther 2, 709-735; Paul, S. B. et al. (2007) Oncology 72, Suppl. 1, 117-123). However, this single marker is low in sensitivity and often has false positive results, such as AFP in patients with large numbers of cirrhosis. In addition, serum AFP can only detect 60% of liver cancer patients. Therefore, it is important to find new molecular markers for early diagnosis of HCC and to develop sensitive detection methods.

巴賽隆納臨床肝癌(BCLC)分期(Llovet,J.M.(2003)Lancet 362,1907-1917)是最近幾年出現的，用於HCC患者的臨床分期。BCLC分期將腫瘤的發展階段與推薦的治療策略聯繫起來，並定義了每個腫瘤分期的護理標準(Llovet,J.M.(2008)J Natl Cancer Inst 100,698-711)。極早期HCC患者(0期)最適合做手術切除。早期HCC患者(A期)最適合做放療(肝腫瘤切除、肝移植或局部消融)。中期HCC患者(B期)適合做肝動脈化學栓塞(TACE)。而中晚期HCC患者(C期)可用索拉菲尼治療。終末期(D期)患者可接受對症治療。 The clinical stage of clinical liver cancer (BCLC) in Paleron (Llovet, J.M. (2003) Lancet 362, 1907-1917) has been in recent years for clinical staging of patients with HCC. BCLC staging links the stage of tumor development to the recommended treatment strategy and defines the standard of care for each tumor stage (Llovet, J.M. (2008) J Natl Cancer Inst 100, 698-711). Very early HCC patients (stage 0) are best for surgical resection. Early HCC patients (stage A) are best for radiotherapy (liver tumor resection, liver transplantation or local ablation). Patients with intermediate HCC (stage B) are eligible for hepatic arterial chemoembolization (TACE). Patients with advanced HCC (C phase) can be treated with sorafenib. Patients with end stage (D) can receive symptomatic treatment.

許多診斷檢驗因受限於它們都是根據僅以單一的分子標誌的分析之事實，所以偵測的可靠性及/或正確性有可能會被影響。又，單一的分子標誌通常不能詳細地預測潛伏期、癌化過程及其相關。因此，目前仍持續需要一種鑑定用之替代性分子標誌及分析方式以克服該等限制。 Many diagnostic tests are limited by the fact that they are based on analysis with only a single molecular marker, so the reliability and/or correctness of the detection may be affected. Moreover, a single molecular marker usually cannot predict latency, cancerization, and its correlation in detail. Therefore, there is still a continuing need for an alternative molecular marker and analytical method for identification to overcome these limitations.

microRNA(miRNA)是一類小的內源性非編碼RNA分子，大小為20~25個核苷酸(nt)，這些小的miRNA通常靶向一個或者多個mRNA，通過翻譯水準的抑制或斷裂靶標mRNAs而調節基因的表現。它們大約在10年前被發現，已被認為在細胞發育、分化、增殖和凋亡中發揮重要作用(Bartel,D.P.(2004)Cell 116,281-297,Ambros,V.(2004) Nature 431,350-355；He,L.et al.(2004)Nat Rev Genet 5,522-531)。miRNA比mRNA在作為腫瘤生物標誌物方面具有更大的優勢，因為它們在體外非常穩定(Lu,J.et al.,(2005)Nature 435,834-838；Lim,L.P.et al.,(2005)Nature 433,769-773)。 MicroRNAs (miRNAs) are small, endogenous, non-coding RNA molecules ranging in size from 20 to 25 nucleotides (nt). These small miRNAs typically target one or more mRNAs, through translational level of inhibition or cleavage of targets. mRNAs regulate the expression of genes. They were discovered about 10 years ago and have been thought to play an important role in cell development, differentiation, proliferation and apoptosis (Bartel, D.P. (2004) Cell 116, 281-297, Ambros, V. (2004) Nature 431, 350-355; He, L. et al. (2004) Nat Rev Genet 5, 522-531). miRNAs have greater advantages over mRNA as tumor biomarkers because they are very stable in vitro (Lu, J. et al., (2005) Nature 435, 834-838; Lim, LP et al., (2005) Nature. 433, 769-773).

miRNA是從初級轉錄物(pri-miRNA)產生的，pri-miRNA被RNase III Drosha加工為含莖-環結構的前體miRNA(pre-miRNA)。接著，在Dicer(一種RNase III)的作用下，髮夾狀的pre-miRNA在細胞質中進一步被切割產生成熟的miRNA，這些成熟的miRNA與其他蛋白質一起組成miRNA-蛋白質複合體(miRNP)。miRNA引導miRNP到達它們的靶mRNA，在此它們發揮各自的功能(綜述參見例如Bartel,D.P.(2004)Cell 23,281-292；He,L.and Hannon,G.J.(2004)Nat.Rev.Genet.5,522-531)。 miRNAs are produced from primary transcripts (pri-miRNAs), which are processed by RNase III Drosha into precursor miRNAs (pre-miRNAs) containing stem-loop structures. Next, under the action of Dicer (an RNase III), the hairpin-like pre-miRNA is further cleaved in the cytoplasm to produce mature miRNAs, which together with other proteins constitute a miRNA-protein complex (miRNP). miRNAs direct miRNPs to their target mRNAs where they exert their respective functions (for review see, for example, Bartel, DP (2004) Cell 23, 281-292; He, L. and Hannon, GJ (2004) Nat. Rev. Genet. 5, 522- 531).

根據miRNA與其靶mRNA之間的互補性程度，miRNA可以指導不同的調節過程。那些與miRNA高度互補的靶mRNA通過與RNA干擾(RNAi)相同的機制被特異降解。因此，在這種情況下，miRNA是擔當小干擾RNA(siRNA)的角色；與miRNA互補性較低的靶mRNA被引導進入細胞降解途徑，或者在蛋白質轉譯上平上被阻遏而mRNA水準不受影響。但是，目前miRNA抑制其靶mRNA轉譯的機制尚有爭議。 Depending on the degree of complementarity between the miRNA and its target mRNA, the miRNA can direct different regulatory processes. Target mRNAs that are highly complementary to miRNAs are specifically degraded by the same mechanism as RNA interference (RNAi). Therefore, in this case, the miRNA acts as a small interfering RNA (siRNA); the target mRNA with low complementarity to the miRNA is directed into the cell degradation pathway, or is repressed in protein translation and the mRNA level is not regulated. influences. However, the mechanism by which miRNAs inhibit the translation of their target mRNA is currently controversial.

高通量microRNA定量技術(如microRNA微陣列)，是以即時定量RT-PCR為基礎的microRNA檢測，為研究癌症基因組中microRNA表現譜提供了有力的工具。可獲得的現有資料表明miRNA表現失調可能與某種癌症的發生和/或發展相關。例如，研究表明hsa-miR-15及hsa-miR-16-1均定位在慢性淋巴性白血病(CLL)中缺失的遺傳基因座上，在大約70%的CLL患者中，這兩種microRNA基因缺失或下調。另外，在結腸癌中has-miR-143及has-miR-145表現下調；而miRNA let-7的表現在肺癌中經常降低(Michael,M.Z.et al.(2003)Mol Cancer Res 1,882-891；Mayr,C.et al.(2007)Science 315,1576-1579)。事實上，常見癌症經常存在相關microRNA表現改變，而且microRNA通常定位於與癌症相關的基因組區域，因此可以推測miRNA可能發揮著抑癌基因和癌基因的雙重作用(Esquela-Kerscher,A.and Slack,F.J(2006)Nat Rev Cancer 6,259-269；Calin,G.A.and Croce,C.M.(2007)J Clin Invest 117,2059-2066；Blenkiron,C.and Miska,E.A.(2007)Hum Mol Genet 16,R106-R113)。已證實的microRNA在人類癌症中的異常表現更突出了它們作為診斷和預後生物標誌物的潛在應用價值。 High-throughput microRNA quantification techniques, such as microRNA microarrays, provide real-time quantitative RT-PCR-based microRNA detection and provide a powerful tool for studying microRNA expression profiles in cancer genomes. Available available data suggest that dysregulation of miRNA may be associated with the development and/or development of certain cancers Related. For example, studies have shown that both hsa-miR-15 and hsa-miR-16-1 are localized in the deleted locus in chronic lymphocytic leukemia (CLL), and in about 70% of CLL patients, the two microRNA genes are deleted. Or down. In addition, has-miR-143 and has-miR-145 are down-regulated in colon cancer; while the expression of miRNA let-7 is often reduced in lung cancer (Michael, MZ et al. (2003) Mol Cancer Res 1, 882-891; Mayr , C. et al. (2007) Science 315, 1576-1579). In fact, common cancers often have associated changes in microRNA expression, and microRNAs are often localized in cancer-associated genomic regions, so it is speculated that miRNAs may play a dual role as tumor suppressor genes and oncogenes (Esquela-Kerscher, A. and Slack, FJ (2006) Nat Rev Cancer 6, 259-269; Calin, GA and Croce, CM (2007) J Clin Invest 117, 2059-2066; Blenkiron, C. and Miska, EA (2007) Hum Mol Genet 16, R106-R113) . The aberrant performance of proven microRNAs in human cancers highlights their potential utility as diagnostic and prognostic biomarkers.

迄今為止，已有學者報導了人HCC中microRNA表現譜(Murakami,Y.et al.(2006)Oncogene 25,2537-2545；Li,W.et al.(2008)Int J Cancer 123,1616-1622；Huang,Y.S.et al.(2008)Hepatology 23,87-94；Ladeiro,Y.et al.(2008)Hepatology 47,1955-1963；Jiang,J.et al.(2008)Clin Cancer Res 14,419-427)。這些研究均表明，與正常肝細胞或組織相比，特定的microRNA在惡性細胞或組織中存在異常表現。因此，它們有助於更深刻理解腫瘤惡性轉化和發展的過程。 To date, scholars have reported microRNA expression profiles in human HCC (Murakami, Y. et al. (2006) Oncogene 25, 2537-2545; Li, W. et al. (2008) Int J Cancer 123, 1616-1622 ; Huang, YSet al. (2008) Hepatology 23, 87-94; Ladeiro, Y. et al. (2008) Hepatology 47, 1955-1963; Jiang, J. et al. (2008) Clin Cancer Res 14, 419-427 ). These studies have shown that specific microRNAs have abnormal expression in malignant cells or tissues compared to normal hepatocytes or tissues. Therefore, they contribute to a deeper understanding of the process of malignant transformation and progression of the tumor.

在諸多類型的標本中，由於血液容易獲取，臨床操作簡單、創傷小，患者所受的風險及痛苦小，被認為最適合用於篩選高危人群，以其早期發現、診斷和治療腫瘤患者。來源於腫瘤的microRNA已被證明在人類血漿或血清中以非常穩定的形式存在，免受內源RNase酶活性的影響。這些在血清或血漿中的來源於腫瘤的microRNA足以被檢測到，可以作為腫瘤生物標誌物。此外，由於血漿和血清的microRNA水準密切相關，血漿或血清中的microRNA都可以作為腫瘤診斷標誌物，而應用於臨床(Mitchell,P.S.et al.(2008)Proc Natl Acad Sci USA 105,10513-10518；Gilad,S.et al.(2008)PLOS ONE 3,e3148；Chen,X.et al.(2008)Cell Res 18,997-1006)。 In many types of specimens, because the blood is easy to obtain, the clinical operation is simple, the trauma is small, and the risk and pain of the patient are small, which is considered to be most suitable. It is used to screen high-risk groups for early detection, diagnosis and treatment of cancer patients. Tumor-derived microRNAs have been shown to exist in very stable forms in human plasma or serum, protected from endogenous RNase enzyme activity. These tumor-derived microRNAs in serum or plasma are sufficient to be detected and can serve as tumor biomarkers. In addition, due to the close correlation between plasma and serum microRNA levels, microRNAs in plasma or serum can be used as tumor diagnostic markers for clinical use (Mitchell, PSet al. (2008) Proc Natl Acad Sci USA 105, 10513-10518 Gilad, S. et al. (2008) PLOS ONE 3, e3148; Chen, X. et al. (2008) Cell Res 18, 997-1006).

最近，有三個研究報導了HCC患者中的血清microRNA。Qu等(Qu,KZ.et al(2011)J Clin Gastroenterol 45：355-60)研究了血清hsa-miR-16、hsa-miR-195以及hsa-miR-196a在283個樣本上的診斷價值，發現hsa-miR-16具有最好的診斷效能，靈敏度和特異性分別為72.1%和88.8%。Xu等(Xu,J.et al(2011)Molecular Carcinogenesis 50：136-42)發現了血液中hsa-miR-21、hsa-miR-122和hsa-miR-223是區分HCC和健康個體的潛在標誌物。然而，這些microRNA不能區分HCC和B型肝炎患者。Li等(Li,LM.et al(2010)Cancer Res 70,9798-807)報導了血清microRNA顯著的診斷價值，hsa-miR-375的靈敏度和特異性分別為96%和100%，hsa-miR-375、hsa-miR-25以及hsa-let-7f聯合應用後，靈敏度和特異性分別為97.9%和99.1%。以上結果提示，血清microRNA診斷HCC是可行的，但是這些研究存在諸多缺陷，如用於篩選的microRNA數目有限，樣本量小或缺少獨立的驗證。因此，仍然有必要在HCC患者的血漿或血清中發現有診斷價值的microRNA生物標誌物。通過多個microRNA生物標誌物聯合應用可以快速、準確、低成本的早期診斷HCC患者。 Recently, three studies have reported serum microRNAs in HCC patients. Qu et al (Qu, KZ. et al (2011) J Clin Gastroenterol 45: 355-60) studied the diagnostic value of serum hsa-miR-16, hsa-miR-195 and hsa-miR-196a in 283 samples. hsa-miR-16 was found to have the best diagnostic efficacy with sensitivity and specificity of 72.1% and 88.8%, respectively. Xu et al (Xu, J. et al (2011) Molecular Carcinogenesis 50: 136-42) found that hsa-miR-21, hsa-miR-122 and hsa-miR-223 in the blood are potential markers for distinguishing between HCC and healthy individuals. Things. However, these microRNAs are incapable of distinguishing between patients with HCC and hepatitis B. Li et al. (Li, LM. et al (2010) Cancer Res 70, 9798-807) reported significant diagnostic value of serum microRNAs with sensitivity and specificity of 96% and 100%, hsa-miR, respectively. After the combination of -375, hsa-miR-25 and hsa-let-7f, the sensitivity and specificity were 97.9% and 99.1%, respectively. These results suggest that the diagnosis of HCC by serum microRNA is feasible, but these studies have many drawbacks, such as limited number of microRNAs for screening, small sample size or lack of independent validation. Therefore, there must still be Diagnostic microRNA biomarkers are found in the plasma or serum of HCC patients. Early diagnosis of HCC patients can be performed quickly, accurately, and at low cost through the combination of multiple microRNA biomarkers.

本發明的第一個目的是提供一個新的HCC(特別是早期肝細胞癌BCLC 0期和A期)的診斷標誌物，從而提供一種新的HCC診斷方法。 A first object of the present invention is to provide a diagnostic marker for a novel HCC (particularly early stage hepatocellular carcinoma BCLC stage 0 and A) to provide a new diagnostic method for HCC.

本發明的第二個目的是提供一種用於診斷HCC(特別是早期肝細胞癌BCLC 0期和A期)的套組。 A second object of the present invention is to provide a kit for diagnosing HCC (particularly early stage hepatocellular carcinoma BCLC stage 0 and stage A).

本發明的第三個目的是提供一種用於區分HCC患者的血漿和健康人的血漿的套組。 A third object of the present invention is to provide a kit for distinguishing plasma of a HCC patient from plasma of a healthy person.

本發明的第四個目的是提供一種用於區分HCC患者的血漿與慢性B型肝炎患者的血漿的套組。 A fourth object of the present invention is to provide a kit for distinguishing plasma of a patient with HCC from plasma of a patient with chronic hepatitis B.

本發明的第五個目的是提供一種用於區分HCC患者的血漿與肝硬化患者的血漿的套組。 A fifth object of the present invention is to provide a kit for distinguishing plasma from patients with cirrhosis of HCC patients.

本發明的第六個目的是提供一種確定肝癌診斷標誌物的方法。 A sixth object of the present invention is to provide a method for determining a diagnostic marker for liver cancer.

這些及其它的目的通過以下描述將變得清楚，它們由獨立項的主題實現。本發明的一些較佳實施方案由附屬項的主題限定。 These and other objects will become apparent from the following description, which is achieved by the subject matter of the individual. Some preferred embodiments of the invention are defined by the subject matter of the dependent items.

在第一方面，本發明公開了一種HCC診斷標誌物，由多種核酸分子組成，每種核酸分子編碼至少一個microRNA序列。 In a first aspect, the invention features an HCC diagnostic marker consisting of a plurality of nucleic acid molecules, each nucleic acid molecule encoding at least one microRNA sequence.

在較佳的實施方案中，所述的多種核酸分子包含至少一種編碼至少一個microRNA序列的核酸分子，其在至少一種靶血漿中與在至少一種對照血漿中差異表現。 In a preferred embodiment, the plurality of nucleic acid molecules comprise at least one nucleic acid molecule encoding at least one microRNA sequence that is differentially expressed in at least one target plasma and in at least one control plasma.

在更佳的實施方案中，所述的在至少一種靶血漿中與在至少一種對照血漿中差異表現的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼microRNA序列的核酸分子，其在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調，以及至少一種編碼microRNA序列的核酸分子，其在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調。 In a further embodiment, the at least one nucleic acid molecule encoding at least one microRNA sequence that differs in at least one target plasma and in at least one control plasma is selected from the group consisting of at least one nucleic acid molecule encoding a microRNA sequence, The performance in at least one target plasma is up-regulated compared to the performance in at least one control plasma, and at least one nucleic acid molecule encoding a microRNA sequence that exhibits in at least one target plasma and in at least one control plasma Than being lowered.

特佳地，所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-801、hsa-miR-192或hsa-miR-21的核酸分子；且所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-122、hsa-miR-26a、hsa-miR-27a或hsa-miR-223的核酸分子。 Particularly preferably, the at least one nucleic acid molecule encoding at least one microRNA sequence that is up-regulated in at least one target plasma compared to the expression in at least one control plasma is selected from the group consisting of at least one encoding hsa-miR-801, a nucleic acid molecule of hsa-miR-192 or hsa-miR-21; and said at least one nucleic acid encoding at least one microRNA sequence that is down-regulated in at least one target plasma compared to expression in at least one control plasma The molecule is selected from at least one nucleic acid molecule encoding hsa-miR-122, hsa-miR-26a, hsa-miR-27a or hsa-miR-223.

在較佳的實施方案中，所述的多種核酸分子還包含至少一種編碼至少一個microRNA序列的核酸分子，其在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比不變。 In a preferred embodiment, the plurality of nucleic acid molecules further comprise at least one nucleic acid molecule encoding at least one microRNA sequence that is unchanged in at least one target plasma as compared to the performance in at least one control plasma.

在更佳的實施方案中，所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比不變的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-1228的核酸分子。 In a more preferred embodiment, the at least one nucleic acid molecule encoding at least one microRNA sequence that is invariant in at least one target plasma as compared to the performance in at least one control plasma is selected from at least one encoding hsa- Nucleic acid molecule of miR-1228.

在最佳的實施方案中，所述的多種核酸分子包含編碼hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a、hsa-miR-801以及hsa-miR-1228 的八個核酸分子的組合。 In a most preferred embodiment, the plurality of nucleic acid molecules comprises hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR- 27a, hsa-miR-801 and hsa-miR-1228 A combination of eight nucleic acid molecules.

在更佳的實施方案中，所述的至少一種對照血漿來自健康個體、慢性B型肝炎患者或肝硬化患者。 In a more preferred embodiment, the at least one control plasma is from a healthy individual, a chronic hepatitis B patient, or a cirrhotic patient.

在較佳的實施方案中，所述的HCC為早期HCC(BCLC 0期和A期)。 In a preferred embodiment, the HCC is an early HCC (BCLC Phase 0 and Phase A).

在第二方面，本發明公開了一種HCC診斷套組，其包含一種HCC診斷標誌物，所述的HCC診斷標誌物，由多種核酸分子組成，每種核酸分子編碼至少一個microRNA序列。 In a second aspect, the invention features an HCC diagnostic kit comprising an HCC diagnostic marker, the HCC diagnostic marker, consisting of a plurality of nucleic acid molecules, each nucleic acid molecule encoding at least one microRNA sequence.

特佳地，所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-801、hsa-miR-192或hsa-miR-21的核酸分子；且所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-122、 hsa-miR-26a,hsa-miR-27a或hsa-miR-223的核酸分子。 Particularly preferably, the at least one nucleic acid molecule encoding at least one microRNA sequence that is up-regulated in at least one target plasma compared to the expression in at least one control plasma is selected from the group consisting of at least one encoding hsa-miR-801, a nucleic acid molecule of hsa-miR-192 or hsa-miR-21; and said at least one nucleic acid encoding at least one microRNA sequence that is down-regulated in at least one target plasma compared to expression in at least one control plasma The molecule is selected from at least one of the codes hsa-miR-122, A nucleic acid molecule of hsa-miR-26a, hsa-miR-27a or hsa-miR-223.

在最佳的實施方案中，所述的多種核酸分子包含編碼hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a、hsa-miR-801以及hsa-miR-1228的八個核酸分子的組合。所述的套組還包含一個回歸模型，如下： logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209*hsa-miR-801 In a most preferred embodiment, the plurality of nucleic acid molecules comprises hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR- A combination of 27a, hsa-miR-801, and eight nucleic acid molecules of hsa-miR-1228. The set also contains a regression model as follows: Logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a- 0.3542*hsa-miR-27a+0.209*hsa-miR-801

其中，hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a以及hsa-miR-801的表現水準是以hsa-miR-1228為內源性對照檢測得到，模型中的logit(p=HCC)值在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調。 Among them, the performance levels of hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR-27a and hsa-miR-801 are hsa- miR-1228 was detected as an endogenous control and the logit (p=HCC) values in the model were up-regulated in at least one target plasma compared to the performance in at least one control plasma.

在第三方面，本發明公開了一種用於區分至少一個HCC患者的血漿和至少一個健康個體的血漿的套組，其包含一種HCC診斷標誌物，所述的HCC診斷標誌物，由多種核酸分子組成，每種核酸分子編碼至少一個microRNA序列，所述的多種核酸分子包含至少一種編碼至少一個microRNA序列的核酸分子，其在至少一種靶血漿中與在至少一種對照血漿中差異表現，所述的至少一種對照血漿來自健康個體。 In a third aspect, the present invention discloses a kit for distinguishing plasma of at least one HCC patient from plasma of at least one healthy individual, comprising an HCC diagnostic marker, said HCC diagnostic marker, comprising a plurality of nucleic acid molecules Composition, each nucleic acid molecule encoding at least one microRNA sequence, the plurality of nucleic acid molecules comprising at least one nucleic acid molecule encoding at least one microRNA sequence that differs in at least one target plasma and in at least one control plasma, said At least one control plasma is from a healthy individual.

特佳地，所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-122、hsa-miR-801、hsa-miR-192或hsa-miR-21的核酸分子；且所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-26a,hsa-miR-27a或hsa-miR-223的核酸分子。 Particularly preferably, the at least one nucleic acid molecule encoding at least one microRNA sequence that is up-regulated in at least one target plasma compared to the expression in at least one control plasma is selected from the group consisting of at least one encoding hsa-miR-122, a nucleic acid molecule of hsa-miR-801, hsa-miR-192 or hsa-miR-21; and said at least one encoding that is downregulated in at least one target plasma compared to performance in at least one control plasma The nucleic acid molecule of at least one microRNA sequence is selected from at least one nucleic acid molecule encoding hsa-miR-26a, hsa-miR-27a or hsa-miR-223.

在第四方面，本發明公開了一種用於區分至少一個HCC患者的血漿和至少一個慢性B型肝炎的血漿的套組，其包含一種HCC診斷標誌物，所述的HCC診斷標誌物，由多種核酸分子組成，每種核酸分子編碼至少一個microRNA序列，所述的多種核酸分子包含至少一種編碼至少一個microRNA序列的核酸分子，其在至少一種靶血漿中與在至少一種對照血漿中差異表現，所述的至少一種對照血漿來自慢性B型肝炎患者。 In a fourth aspect, the present invention discloses a kit for distinguishing plasma of at least one HCC patient from at least one chronic hepatitis B virus, comprising an HCC diagnostic marker, said HCC diagnostic marker, Consisting of a plurality of nucleic acid molecules, each nucleic acid molecule encoding at least one microRNA sequence comprising at least one nucleic acid molecule encoding at least one microRNA sequence that differs in at least one target plasma and in at least one control plasma The at least one control plasma is from a chronic hepatitis B patient.

在更加較佳的實施方案中，所述的在至少一種靶血漿中與在至少一種對照血漿中差異表現的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼microRNA序列的核酸分子，其在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調，以及至少一種編碼microRNA序列的核酸分子，其在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調。 In a more preferred embodiment, the at least one nucleic acid molecule encoding at least one microRNA sequence that differs in at least one target plasma and in at least one control plasma is selected from at least one nucleic acid molecule encoding a microRNA sequence. The performance in at least one target plasma is up-regulated compared to the performance in at least one control plasma, and the performance of at least one nucleic acid molecule encoding a microRNA sequence in at least one target plasma and in at least one control plasma Compared to being down.

特佳地，所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-801或hsa-miR-21的核酸分子；且所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-122、hsa-miR-192、hsa-miR-26a、hsa-miR-27a或hsa-miR-223的核酸分子。 Particularly preferably, the at least one nucleic acid molecule encoding at least one microRNA sequence that is up-regulated in at least one target plasma compared to the expression in at least one control plasma is selected from at least one of the encoding hsa-miR-801 or a nucleic acid molecule of hsa-miR-21; and said at least one nucleic acid molecule encoding at least one microRNA sequence that is down-regulated in at least one target plasma as compared to expression in at least one control plasma is selected from at least one encoding A nucleic acid molecule of hsa-miR-122, hsa-miR-192, hsa-miR-26a, hsa-miR-27a or hsa-miR-223.

在較佳的實施方案中，所述的多種核酸分子還包含至少一種編碼至少一個microRNA序列的核酸分子，其在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比不變。 In a preferred embodiment, the plurality of nucleic acid molecules further comprise at least one nucleic acid molecule encoding at least one microRNA sequence, the performance in at least one target plasma and the expression in at least one control plasma The ratio is unchanged.

在第五方面，本發明公開了一種用於區分至少一個HCC患者的血漿和至少一個肝硬化患者的血漿的套組，其包含一種HCC診斷標誌物，所述的HCC診斷標誌物，由多種核酸分子組成，每種核酸分子編碼至少一個microRNA序列，所述的多種核酸分子包含至少一種編碼至少一個microRNA序列的核酸分子，其在至少一種靶血漿中與在至少一種對照血漿中差異表現，所述的至少一種對照血漿來自肝硬化患者。 In a fifth aspect, the present invention discloses a kit for distinguishing plasma of at least one HCC patient from plasma of at least one cirrhotic patient, comprising an HCC diagnostic marker, said HCC diagnostic marker, comprising a plurality of nucleic acids a molecular composition, each nucleic acid molecule encoding at least one microRNA sequence, the plurality of nucleic acid molecules comprising at least one nucleic acid molecule encoding at least one microRNA sequence in at least one target plasma There is less variation in the control plasma, and at least one of the control plasmas is from a patient with cirrhosis.

特佳地，所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-801的核酸分子；且所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-26a、hsa-miR-27a或hsa-miR-223的核酸分子。 Particularly preferably, the at least one nucleic acid molecule encoding at least one microRNA sequence that is up-regulated in at least one target plasma compared to the expression in at least one control plasma is selected from at least one of the codes encoding hsa-miR-801. a nucleic acid molecule; and the at least one nucleic acid molecule encoding at least one microRNA sequence that is down-regulated in at least one target plasma compared to the expression in at least one control plasma is selected from the group consisting of at least one encoding hsa-miR-122, A nucleic acid molecule of hsa-miR-192, hsa-miR-21, hsa-miR-26a, hsa-miR-27a or hsa-miR-223.

在更加較佳的實施方案中，所述的在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比不變的至少一種編碼至少一個microRNA序列的核酸分子選自至少一種編碼hsa-miR-1228的核酸分子。 In a more preferred embodiment, said performance in at least one target plasma is at least constant compared to performance in at least one control plasma A nucleic acid molecule encoding at least one microRNA sequence is selected from at least one nucleic acid molecule encoding hsa-miR-1228.

在最佳的實施方案中，所述的多種核酸分子包含編碼hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a、hsa-miR-801以及hsa-miR-1228的八個核酸分子的組合。所述的套組還包含一個回歸模型，如下： logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209*hsa-miR-80 In a most preferred embodiment, the plurality of nucleic acid molecules comprises hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR- A combination of 27a, hsa-miR-801, and eight nucleic acid molecules of hsa-miR-1228. The set also contains a regression model as follows: Logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a- 0.3542*hsa-miR-27a+0.209*hsa-miR-80

在第六方面，本發明公開了一種確定肝癌診斷標誌物的方法，包括： (a)在一個或多個靶血漿中確定多個核酸分子的表現水準，每個核酸分子編碼至少一個microRNA序列； (b)在一個或多個對照血漿中確定上述多個核酸分子的表現水準； (c)通過對比在步驟(a)和(b)中獲得的多個核酸分子的相應表現水準，來從所述多個核酸分子中鑑定出在所述的靶血漿和所述的對照血漿中差異表現的一個或多個核酸分子，將在所述的靶血漿和所述的對照血漿中差異表現的一個或多個核酸分子作為肝癌診斷標誌物。 In a sixth aspect, the invention discloses a method for determining a diagnostic marker for liver cancer, comprising: (a) determining the performance level of a plurality of nucleic acid molecules in one or more target plasmas, each nucleic acid molecule encoding at least one microRNA sequence; (b) determining the performance level of said plurality of nucleic acid molecules in one or more control plasmas; (c) identifying from said plurality of nucleic acid molecules in said target plasma and said control plasma by comparing the respective expression levels of the plurality of nucleic acid molecules obtained in steps (a) and (b) One or more cores of differential performance The acid molecule is one or more nucleic acid molecules that are differentially expressed in the target plasma and the control plasma as a diagnostic marker for liver cancer.

本發明的其他實施方案將從以下詳細描述中變得明瞭。 Other embodiments of the invention will be apparent from the following detailed description.

本發明以如下出乎意料的發現為基礎，即基於具有高診斷準確度的肝癌診斷標誌物，HCC可以被可靠地鑑定。典型地，這裡定義的肝癌診斷標誌物包含被上調和下調的人類microRNA。更具體地，通過分析血漿中整體microRNA表現模式和/或單個microRNA的表現水準，所述的肝癌診斷標誌物使得HCC能夠在早期疾病狀態得到檢測，HCC患者的血漿和健康個體、慢性B型肝炎患者及肝硬化患者的血漿能夠得到區分。 The present invention is based on the unexpected findings that HCC can be reliably identified based on liver cancer diagnostic markers with high diagnostic accuracy. Typically, a liver cancer diagnostic marker as defined herein comprises a human microRNA that is up-regulated and down-regulated. More specifically, by analyzing the overall microRNA expression pattern in plasma and/or the performance level of a single microRNA, the liver cancer diagnostic marker enables HCC to be detected in an early disease state, plasma and healthy individuals of HCC patients, chronic hepatitis B The plasma of patients and patients with cirrhosis can be distinguished.

以下舉例說明的本發明可以適當地在缺少未在本文中特別揭示的任何一個或多個元素或限制的條件下實施。 The invention exemplified below can be suitably carried out in the absence of any one or more of the elements or limitations not specifically disclosed herein.

本發明將根據特定的即時方式並參照附圖加以描述，但是本發明不受其限制，僅受到申請專利範圍限制。所描述的附圖僅是示意性的，被認為是非限制性的。 The present invention will be described in terms of a specific instant manner and with reference to the accompanying drawings, but the invention is not limited thereto, but only by the scope of the claims. The drawings described are only schematic and are considered to be non-limiting.

當術語“包含”被用在本發明說明書和申請專利範圍中時，其不排除其它元素或步驟。為了實現本發明的目的，術語“由...組成”被認為是術語“包含”的較佳實施方案。如果在下文中某一組被定義為包含至少一定數目的實施方案，這也應被理解為揭示了一個較佳地僅由這些實施方案組成的組。 When the term "comprising" is used in the context of the present specification and claims, it does not exclude other elements or steps. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising." If a group is defined below to include at least a certain number of embodiments, this should also be understood to reveal a group that preferably consists only of these embodiments.

除非特別聲明，在提及單數形式名詞時使用的不定冠詞或定冠詞例如“一個”或“一種”，“所述”，包括該名詞的複數形式。 Unless otherwise stated, an indefinite or definite article such as "a" or "an" Number form.

本發明中的術語“大約”表示本領域技術人員能夠理解的仍可保證論及特徵的技術效果的準確度區間。該術語通常表示偏離指示數值的±10%，較佳±5%。 The term "about" in the present invention denotes an accuracy interval that can be understood by those skilled in the art to still assure the technical effects of the features. The term generally means ±10%, preferably ±5%, of the indicated value.

此外，說明書和申請專利範圍中的術語第一、第二、第三、(a)、(b)、(c)以及諸如此類，是用於區分相似的元素，不是描述順序或時間次序必須的。應理解，如此應用的術語在適當的環境下可互換，並且本發明描述的實施方案能以不同於本文描述或舉例說明的其它順序實施。 In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not required to describe the order or chronological order. It is understood that the terms so applied are interchangeable under appropriate circumstances and that the embodiments described herein can be implemented in other sequences than those described or illustrated herein.

術語在其所應用的語境中的進一步定義將在下文中給出。 Further definitions of terms in the context in which they are applied will be given below.

以下術語或定義僅僅是為了幫助理解本發明而提供。這些定義不應被理解為具有小於本領域技術人員所理解的範圍。 The following terms or definitions are provided solely to assist in understanding the invention. These definitions should not be construed as having a range less than those understood by those skilled in the art.

本文所用的術語“癌症”(也稱為“癌”)通常表示任何類型的惡性贅生物，即與未受影響的(健康的)野生型對照細胞相比顯示或具有發生癌特徵傾向的靶細胞的任何形態學和/或生理學改變(基於基因重程式設計)。這種改變可涉及細胞大小和形狀(變大或變小)、細胞增殖(細胞數增加)、細胞分化(生理學狀態變化)、凋亡(程式性細胞死亡)或細胞存活。 The term "cancer" (also referred to as "cancer") as used herein generally refers to any type of malignant neoplasm, ie a target cell that exhibits or has a predisposition to develop a cancerous characteristic compared to an unaffected (healthy) wild type control cell. Any morphological and/or physiological changes (based on gene reprogramming). Such alterations may involve cell size and shape (greater or small), cell proliferation (increased cell number), cell differentiation (physiological state changes), apoptosis (programmed cell death), or cell survival.

本文所用的術語“肝細胞”是指肝臟的細胞。因此，術語“肝癌”是指在肝臟中的癌性生長。 The term "hepatocyte" as used herein refers to a cell of the liver. Thus, the term "liver cancer" refers to cancerous growth in the liver.

肝癌的最常見類型是肝細胞癌(也稱作肝癌，通常縮寫為HCC)。本文所用的術語“肝細胞癌”表示肝臟原發惡性腫瘤。大多數HCC繼發於病毒性肝炎感染(B型肝炎或C型肝炎)或者肝硬化(酒精中毒是導致肝硬化的最常見因素)。在肝炎不是地方病的國家中，大多數肝臟惡性癌症不是原發性HCC，而是從機體其它部位例如結腸的癌症轉移(播散)。HCC的治療選擇和預後依賴於許多因素，但是特別依賴於腫瘤大小和分期。通常結果不佳是因為僅10%~20%的HCC能夠通過手術徹底切除。如果癌症不能被完全切除，則該疾病通常在3-6個月內是致命的。 The most common type of liver cancer is hepatocellular carcinoma (also known as liver cancer, often abbreviated as HCC). The term "hepatocellular carcinoma" as used herein refers to a primary malignant tumor of the liver. Most HCC is secondary to viral hepatitis infection (Hepatitis B or Hepatitis C) or cirrhosis (alcoholism is the most common cause of cirrhosis). In countries where hepatitis is not endemic, most hepatic malignancies are not primary HCC, but cancer metastasis (dispersion) from other parts of the body, such as the colon. The treatment options and prognosis of HCC depend on many factors, but are particularly dependent on tumor size and stage. Usually the result is poor because only 10% to 20% of HCC can be completely removed by surgery. If the cancer cannot be completely removed, the disease is usually fatal within 3-6 months.

與任何其它癌症一樣，HCC在存在引起細胞高速複製和/或導致細胞避免凋亡的細胞突變時發生。特別地，B型肝炎和/或C型肝炎的慢性病毒感染能夠通過反覆引起機體自身免疫系統攻擊肝細胞(其中一些被病毒感染，其它的僅是旁觀者)而有助於HCC的發生。這種損傷-修復-再損傷的循環可導致修復期間的錯誤，最後將導致癌變，但是目前這種假說更適用於C型肝炎。然而，在B型肝炎中，病毒基因組整合進感染的細胞中是惡性腫瘤發生中最相關的因素。另外，反覆大量飲酒具有相似作用。 As with any other cancer, HCC occurs when there is a mutation in a cell that causes rapid cell replication and/or causes the cell to avoid apoptosis. In particular, chronic viral infections of hepatitis B and/or hepatitis C can contribute to the development of HCC by repeatedly inducing the body's own immune system to attack liver cells, some of which are infected by viruses, others are only bystanders. This injury-repair-re-injury cycle can lead to errors during repair and eventually lead to cancer, but this hypothesis is now more applicable to hepatitis C. However, in hepatitis B, integration of the viral genome into infected cells is the most relevant factor in malignant tumorigenesis. In addition, repeated heavy drinking has a similar effect.

巴賽隆納臨床肝癌(BCLC)分期方案包含5個時期。極早期(0期)包括具有無臨床症狀的單個HCC<2cm的患者。早期(A期)包括具有無臨床症狀的單個或三個HCC<=3 cm的患者。中期(B期)包括具有無臨床症狀的多個HCC結節的患者。進展期(C期)包括具有臨床症狀的腫瘤和/或侵襲性腫瘤模式(血管侵犯或肝外播散)的患者。末期(D期)包括具有極其令人擔憂的預後的患者。 The Barcelona clinical liver cancer (BCLC) staging program consists of five periods. Very early stage (stage 0) included patients with a single clinical HCC < 2 cm without clinical symptoms. Early (stage A) included patients with no clinical symptoms of single or three HCC <= 3 cm. Mid-term (phase B) includes patients with multiple HCC nodules without clinical symptoms. The progression phase (C phase) includes patients with clinically symptomatic tumors and/or invasive tumor patterns (vascular invasion or extrahepatic dissemination). The terminal phase (D phase) includes patients with an extremely worrying prognosis.

因此，在本發明範圍中，B型肝炎感染或者肝硬化不僅被視為腫瘤病因學的危險因素，而且還是腫瘤發展的早期/中期(即“癌前狀態”)，其與導致(通常為良性)非侵襲性贅生物的過度增殖性組織生長(隨之可發展為惡性腫瘤如 HCC)相關。 Thus, within the scope of the present invention, hepatitis B infection or cirrhosis is not only considered a risk factor for tumor etiology, but also an early/mid-term (ie, "precancerous state") of tumor development, which is associated with (usually benign) Hyperproliferative tissue growth of non-invasive neoplasms (and subsequently develop into malignant tumors such as HCC) related.

這種惡性腫瘤侵襲其它組織並經常發生轉移。惡性細胞通常以進展性和不受控制的生長為特徵。肉眼觀察HCC看起來是結節性或浸潤性腫瘤。結節型腫瘤可以是單發性(具有大體積)或多發性(當發展為硬化併發症時)。腫瘤結節為圓形至橢圓形，邊界清楚但無包膜。彌漫型邊界不清，且浸潤門靜脈，很少浸潤肝靜脈。 This malignant tumor invades other tissues and often metastasizes. Malignant cells are often characterized by progressive and uncontrolled growth. Visual observation of HCC appears to be a nodular or invasive tumor. Nodular tumors can be either single (large volume) or multiple (when developing a complication of cirrhosis). The tumor nodules are round to elliptical with clear boundaries but no envelope. Diffuse borders are unclear and infiltrate the portal vein, rarely infiltrating the hepatic vein.

本發明中採用的哺乳動物靶血漿可以是人或非人類來源的。然而，本發明典型地採用人類血漿進行。本文所用的術語“一種或多種血漿”應被理解為不僅包括個體血漿。本文所用的術語“靶血漿”是指被至少認定是顯示HCC的血漿，而術語“對照血漿”典型地表示從健康個體、慢性B型肝炎患者和肝硬化患者得到的不具有這種癌症特徵的血漿。但是，在一些應用中，例如在不同癌症類型的血漿之間比較時，不具有HCC表型的血漿典型地被認為是“對照血漿”。 The mammalian target plasma employed in the present invention may be of human or non-human origin. However, the invention is typically carried out using human plasma. The term "one or more plasmas" as used herein shall be understood to include not only individual plasma. The term "target plasma" as used herein refers to plasma that is at least identified to be HCC, and the term "control plasma" typically refers to those obtained from healthy individuals, chronic hepatitis B patients, and cirrhotic patients that do not have such cancer characteristics. plasma. However, in some applications, such as when comparing between plasmas of different cancer types, plasma without the HCC phenotype is typically considered "control plasma."

本發明所用的術語“血漿”，是血液中的黃色液體成分，其中全血中的血細胞通常以懸浮狀態存在。它通常占了血液總體積的大約55%。它大部分是水(占體積的90%)，含有溶解的蛋白質、葡萄糖、凝血因子、礦物質離子、激素以及二氧化碳(血漿是***產物運輸的主要途徑)。血漿是通過將一管鮮血在離心機中離心直到血細胞沉在管底，隨後將血漿倒出或抽出的方法來製備。血漿密度約為1025 kg/m³或1.025 kg/l。最近研究表明microRNA在血漿中是穩定的。術語“血漿樣本”指從被檢測的個人或對照中取得的血漿。 The term "plasma" as used in the present invention is a yellow liquid component in blood in which blood cells in whole blood are usually present in a suspended state. It usually accounts for about 55% of the total blood volume. It is mostly water (90% by volume) and contains dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide (plasma is the main route of transport of excreta products). Plasma is prepared by centrifuging a tube of blood in a centrifuge until the blood cells sink to the bottom of the tube, followed by pouring or withdrawing the plasma. The plasma density is approximately 1025 kg/m ³ or 1.025 kg/l. Recent studies have shown that microRNAs are stable in plasma. The term "plasma sample" refers to plasma taken from a person or control being tested.

本發明所用的術語“患者”是指至少被認為患有HCC的人類，而本發明所用的“靶血漿”是指從患者中採集到的血漿。術語“健康個體”典型地一般表示不患有癌症的健康人。本發明所用的“對照血漿”表示從健康個體、慢性B型肝炎患者以及肝硬化患者採集到的血漿。但是，在一些應用中，例如當比較不同的癌症類型時，患有其它癌症類型的個體以及從這些個體採集到的血漿典型地被認為是對照。 The term "patient" as used in the present invention refers to a human at least considered to have HCC, and "target plasma" as used herein refers to plasma collected from a patient. The term "healthy individual" typically refers generally to a healthy person who does not have cancer. As used herein, "control plasma" means plasma collected from healthy individuals, chronic hepatitis B patients, and cirrhotic patients. However, in some applications, such as when comparing different cancer types, individuals with other cancer types and plasma collected from these individuals are typically considered to be controls.

典型地，使用的血漿樣本來源於從被診斷患有HCC的受試者採集的生物試樣。此外，為了確證獲得的資料，對比樣本可以從具有已知疾病狀態的受試者採集。所述的生物學樣本可包括機體組織及液體，如組織、血清、血細胞、痰和尿。另外，生物學樣本可從具有HCC或疑為癌症的個體獲得。另外，所述的樣本如有需要可從獲得的機體組織或液體中提純後作為生物學樣本使用。根據本發明，本發明中核酸標誌物的表現水準在由物件衍生的生物學樣本中確定。 Typically, the plasma sample used is derived from a biological sample taken from a subject diagnosed with HCC. Furthermore, to confirm the data obtained, the comparison sample can be taken from a subject with a known disease state. The biological sample can include body tissues and fluids such as tissues, serum, blood cells, sputum, and urine. Additionally, biological samples can be obtained from individuals with HCC or suspected cancer. In addition, the sample can be used as a biological sample after purification from the obtained body tissues or liquids as needed. According to the present invention, the performance level of the nucleic acid marker of the present invention is determined in a biological sample derived from the article.

在本發明的體外方法中用於檢測的樣本通常應以臨床可接受的方式採集，較佳以核酸(特別是RNA)或蛋白質的方式保存。待分析的樣品典型地來自血液。另外，肝組織或其它類型的樣本也可以使用。樣本，尤其在初步加工後可以合併。但是，也可以使用沒有合併的樣本。 The samples for detection in the in vitro methods of the invention should generally be collected in a clinically acceptable manner, preferably in the form of nucleic acids (especially RNA) or proteins. The sample to be analyzed is typically from blood. In addition, liver tissue or other types of samples can also be used. Samples, especially after initial processing, can be combined. However, it is also possible to use samples that are not merged.

本發明所用的術語“microRNA”(或"miRNA")具有其在本領域中的普通含義(Bartel,D.P.(2004)Cell 23,281-292；He,L.and Hannon,G.J.(2004)Nat Rev Genet 5,522-531)。因此，“microRNA”表示來自遺傳基因位點的RNA分子，其從可以形成局部RNA前體miRNA結構的轉錄物加工而來。成熟miRNA通常長度為20、21、22、23、24或25個核苷酸，儘管其它數目的核苷酸也可存在，例如18、19、26或27個核苷酸。 The term "microRNA" (or "miRNA") as used in the present invention has its ordinary meaning in the art (Bartel, DP (2004) Cell 23, 281-292; He, L. and Hannon, GJ (2004) Nat Rev Genet 5, 522 -531). Thus, "microRNA" refers to an RNA molecule from a genetic locus, Transcription from a transcript that can form a local RNA precursor miRNA structure. Mature miRNAs are typically 20, 21, 22, 23, 24 or 25 nucleotides in length, although other numbers of nucleotides may be present, such as 18, 19, 26 or 27 nucleotides.

miRNA編碼序列具有與側翼基因組序列配對的潛力，使成熟miRNA放置在非完全配對的RNA雙鏈體之內(本文也稱作莖-環或髮夾結構或pre-miRNA)，所述雙鏈體作為從更長的前體轉錄物進行miRNA加工的中間體。這一加工典型地通過分別稱為Drosha和Dicer的兩種特定的內切核酸酶的連續作用而發生。Drosha從初級轉錄物(本文也稱作“pri-miRNA”)產生典型地折疊成髮夾或莖-環結構的miRNA前體(本文也稱作“pre-miRNA”)。採用Dicer方法切割這個miRNA前體可以得到miRNA雙鏈體，其髮夾或莖-環結構的一條臂包含成熟miRNA，另一條臂包含類似大小的節段(通常稱為miRNA*)。所述的miRNA隨後被引導到其靶mRNA以發揮其功能，而miRNA*被降解。另外，miRNA典型地衍生自與預測的蛋白質編碼區不同的基因組節段。 The miRNA coding sequence has the potential to pair with flanking genomic sequences such that the mature miRNA is placed within a non-fully paired RNA duplex (also referred to herein as a stem-loop or hairpin structure or pre-miRNA), the duplex As an intermediate for miRNA processing from longer precursor transcripts. This processing typically occurs through the sequential action of two specific endonucleases, designated Drosha and Dicer, respectively. Drosha produces miRNA precursors (also referred to herein as "pre-miRNAs") that are typically folded into a hairpin or stem-loop structure from a primary transcript (also referred to herein as "pri-miRNA"). The Dicer method is used to cleave this miRNA precursor to obtain a miRNA duplex in which one arm of the hairpin or stem-loop structure contains the mature miRNA and the other arm contains a segment of similar size (commonly referred to as miRNA*). The miRNA is then directed to its target mRNA to perform its function, while the miRNA* is degraded. In addition, miRNAs are typically derived from genomic segments that differ from the predicted protein coding region.

本發明所用的術語“miRNA前體”(或“前體miRNA”或“pre-miRNA”)是指miRNA初級轉錄物的一部分，成熟miRNA從該miRNA初級轉錄物加工得到。典型地，pre-miRNA折疊成穩定的髮夾(即雙鏈體)或莖-環結構。髮夾結構典型地長度為50-80個核苷酸，較佳60-70個核苷酸(將miRNA殘基，與miRNA配對的殘基，及任何間插節段都算在內，但排除更遠端的序列)。 The term "miRNA precursor" (or "precursor miRNA" or "pre-miRNA") as used herein refers to a portion of a miRNA primary transcript from which a mature miRNA is processed. Typically, the pre-miRNA folds into a stable hairpin (ie, duplex) or stem-loop structure. The hairpin structure is typically 50-80 nucleotides in length, preferably 60-70 nucleotides (residing miRNA residues, residues paired with miRNA, and any intervening segments, but excluding More distal sequence).

本發明所用的術語“編碼微RNA序列的核酸分子”是指編碼microRNA(miRNA)的任何核酸分子。因此，該術語不僅指成熟miRNA，也指相應的如上所述的前體miRNA及初級miRNA轉錄物。另外，本發明不限於RNA分子，也包括相應的編碼microRNA的DNA分子，例如通過逆轉錄miRNA序列產生的DNA分子。編碼本發明的microRNA序列的核酸分子典型地編碼單個miRNA序列(即個體miRNA)。但是，也可能這種核酸分子編碼兩個或多個miRNA序列(即兩個或多個miRNA)，例如一個轉錄單位包含在常用調節序列如啟動子或轉錄終止子控制下的兩個或多個miRNA序列。 The term "nucleic acid molecule encoding a microRNA sequence" as used in the present invention means Any nucleic acid molecule encoding a microRNA (miRNA). Thus, the term refers not only to mature miRNAs, but also to corresponding precursor miRNAs and primary miRNA transcripts as described above. In addition, the invention is not limited to RNA molecules, but also includes corresponding DNA molecules encoding microRNAs, such as DNA molecules produced by reverse transcription of miRNA sequences. A nucleic acid molecule encoding a microRNA sequence of the invention typically encodes a single miRNA sequence (ie, an individual miRNA). However, it is also possible that the nucleic acid molecule encodes two or more miRNA sequences (ie two or more miRNAs), eg one transcription unit comprises two or more under the control of commonly used regulatory sequences such as promoters or transcription terminators. miRNA sequence.

本發明所用的術語“編碼microRNA序列的核酸分子”也被理解為包括“有義核酸分子”(即核酸序列(5'→3')匹配或相應於所編碼的miRNA(5'→3')序列的分子)及“反義核酸分子”(即核酸序列互補於所編碼的miRNA(5'→3')序列，或者換句話說，匹配所編碼的miRNA序列的反向互補序列(3'→5')的分子)。本發明所用的術語“互補”是指“反義”核酸分子序列與相應的“有義”核酸分子序列(具有互補於反義序列的序列)形成鹼基對(較佳Watson-Crick鹼基對)的能力。 The term "nucleic acid molecule encoding a microRNA sequence" as used in the present invention is also understood to include "sense nucleic acid molecule" (ie nucleic acid sequence (5'→3') match or corresponding to the encoded miRNA (5'→3'). a sequence of molecules) and an "antisense nucleic acid molecule" (ie, the nucleic acid sequence is complementary to the encoded miRNA (5'→3') sequence, or in other words, matches the reverse complement of the encoded miRNA sequence (3'→ 5') of the molecule). The term "complementary" as used in the present invention means that the sequence of the "antisense" nucleic acid molecule forms a base pair with the corresponding "sense" nucleic acid molecule sequence (having a sequence complementary to the antisense sequence) (preferably Watson-Crick base pair) )Ability.

在本發明範圍內，兩個核酸分子(即“有義”和“反義”分子)可以完全互補，即它們不含有任何鹼基錯配和/或額外的或缺失的核苷酸。或者，兩個分子包含一個或多個鹼基錯配或者在它們的核苷酸總數上不同(由於添加或缺失導致)。較佳地，“互補”核酸分子包含與包含在相應的“有義”核酸分子中的序列顯示完全互補性的至少10個連續核苷酸。 Within the scope of the invention, two nucleic acid molecules (ie, "sense" and "antisense" molecules) may be fully complementary, ie, they do not contain any base mismatches and/or additional or deleted nucleotides. Alternatively, two molecules contain one or more base mismatches or differ in their total number of nucleotides (due to addition or deletion). Preferably, a "complementary" nucleic acid molecule comprises at least 10 contiguous nucleotides that exhibit complete complementarity to a sequence contained in a corresponding "sense" nucleic acid molecule.

因此，包含在本發明的診斷套組中的編碼miRNA序列的多個核酸分子可包括一個或多個“有義核酸分子”和/或一個或多個“反義核酸分子”。有時，所述的診斷套組包括一個或多個“有義核酸分子”(即miRNA序列本身)，所述分子被認為組成了差異表現的miRNA(即分子標誌物)的全體或至少一個子集合，所述差異表現的miRNA是特定情況存在或發生傾向的指征，所述的特定情況在本發明中指HCC。另一方面，當診斷套組包括一個或多個“反義核酸分子”(即與miRNA序列互補的序列)時，所述分子可包含適合用於檢測和/或定量給定樣品中的一個或多個特定(互補)miRNA序列的探針分子(用於進行雜交測定)和/或寡核苷酸引物(例如用於逆轉錄或PCR應用)等。 Thus, a plurality of nucleic acid molecules encoding a miRNA sequence contained in a diagnostic kit of the invention may comprise one or more "sense nucleic acid molecules" and/or one or more "antisense nucleic acid molecules". Sometimes, the diagnostic kit comprises one or more "sense nucleic acid molecules" (ie, the miRNA sequence itself) that are considered to constitute all or at least one of the differentially expressed miRNAs (ie, molecular markers). The set, the differentially expressed miRNA is an indication of the presence or tendency of a particular condition, which in the present invention refers to HCC. In another aspect, when the diagnostic kit comprises one or more "antisense nucleic acid molecules" (ie, sequences complementary to the miRNA sequence), the molecule can comprise one suitable for detecting and/or quantifying one of the given samples or Probe molecules for a plurality of specific (complementary) miRNA sequences (for performing hybridization assays) and/or oligonucleotide primers (for example for reverse transcription or PCR applications) and the like.

在本發明中定義的多個核酸分子可以包含至少2個、至少10個、至少50個、至少100個、至少200個、至少500個、至少1000個、至少10000個或至少100000個核酸分子，每個分子編碼至少一個miRNA序列。 The plurality of nucleic acid molecules defined in the present invention may comprise at least 2, at least 10, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 10,000 or at least 100,000 nucleic acid molecules, Each molecule encodes at least one miRNA sequence.

本發明所用的術語“差異表現”是指特定microRNA在靶血漿中的表現水準與在對照血漿中相比是改變的，所述對照血漿可以是健康個體的血漿或其它類型疾病患者的血漿，其可以是上調(即在靶血漿中microRNA濃度增加)或下調(即在靶血漿中microRNA濃度降低或消失)。換句話說，核酸分子在靶血漿中被啟動至比在對照血漿中更高或更低的水準。 As used herein, the term "differential expression" means that the performance level of a particular microRNA in a target plasma is altered compared to that in a control plasma, which may be the plasma of a patient or a patient of another type of disease in a healthy individual. It may be up-regulated (ie, an increase in microRNA concentration in the target plasma) or down-regulated (ie, the microRNA concentration in the target plasma is reduced or disappeared). In other words, the nucleic acid molecule is initiated in the target plasma to a higher or lower level than in the control plasma.

在本發明範圍內，如果核酸分子在靶血漿和對照血漿中的相應表現水準典型地相差至少5%或至少10%，較佳至少20%或至少25%，最較佳至少30%或至少50%，則該核酸分子被認為是差異表現的。因此，後者的值相應於給定核酸分子在靶血漿中的表現水準與對照細胞相比上調至少1.3倍或至少1.5倍，或反之在靶血漿中的表現水準下調至少0.7倍或至少0.5倍。 Within the scope of the invention, if the corresponding performance level of the nucleic acid molecule in the target plasma and control plasma typically differs by at least 5% or at least 10%, preferably at least 20% or at least 25%, most preferably at least 30% or at least 50. %, then the core Acid molecules are considered to be differentially expressed. Thus, the latter value corresponds to a level of expression of a given nucleic acid molecule in the target plasma that is at least 1.3 fold or at least 1.5 fold higher than the control cell, or vice versa, at least a factor of 0.7 or at least 0.5 fold downregulated in the target plasma.

本發明所用的術語“表現水準”是指特定的miRNA序列從其基因組基因座被轉錄的程度，即miRNA在一個或多個被分析血漿中的濃度。 As used herein, the term "performance level" refers to the extent to which a particular miRNA sequence is transcribed from its genomic locus, ie, the concentration of the miRNA in one or more of the plasma being analyzed.

如上所述，術語“對照血漿”典型地指從不具有HCC表型特徵的個體採集的血漿。但是，在一些應用中，例如當比較不同癌症時，從其它癌症患者採集的血漿典型地被認為是“對照血漿”。 As noted above, the term "control plasma" typically refers to plasma collected from individuals who do not have HCC phenotypic characteristics. However, in some applications, such as when comparing different cancers, plasma collected from other cancer patients is typically considered "control plasma."

表現水準的測定典型地遵循本領域熟知的已建立的標準程式(Sambrook,J.et al.(1989)Molecular Cloning：A Laboratory Manual.2nd Ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY；Ausubel,F.M.et al.(2001)Current Protocols in Molecular Biology.Wiley & Sons,Hoboken,NJ)。所述的測定可以在RNA水準進行，例如使用miRNA特異性探針進行Northern印跡分析，或者在RNA逆轉錄(及選殖)後的DNA水準進行，例如通過定量PCR或即時PCR技術。本發明所用的術語“測定”包括編碼上述至少一個microRNA序列的任何核酸分子的分析。但是，由於pri-miRNA及re-mRNA半生期短，僅典型地測量成熟miRNA的濃度。 Performance level measurements typically follow established standard procedures well known in the art (Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, FM et al. (2001) Current Protocols in Molecular Biology. Wiley & Sons, Hoboken, NJ). Such assays can be performed at the RNA level, for example, using Northern blot analysis using miRNA-specific probes, or at the DNA level following RNA reverse transcription (and colonization), such as by quantitative PCR or real-time PCR techniques. The term "assay" as used in the present invention includes the analysis of any nucleic acid molecule encoding the at least one microRNA sequence described above. However, since the half-life of pri-miRNA and re-mRNA is short, only the concentration of mature miRNA is typically measured.

在特定的實施方案中，在一個給定樣品的若干獨立測量(例如兩次、三次、五次或十次測量)和/或在若干靶血漿或對照血漿內的若干次測量中獲得的表現水準的標準值被用於分析。標準值可以用本領域已知的任何方法獲得。例如，平均值±2 SD(標準差)或平均值±3 SD的範圍可被用作標準值。 In a particular embodiment, performance levels obtained in several independent measurements (eg, two, three, five, or ten measurements) of a given sample and/or in several measurements in several target plasmas or control plasmas Standard value is Used for analysis. Standard values can be obtained by any method known in the art. For example, a range of the mean ± 2 SD (standard deviation) or the mean ± 3 SD can be used as the standard value.

所獲得的靶血漿和對照血漿的表現水準之間的差異可以進一步以對照核酸進行標準化，例如管家基因的表現水準，已知管家基因的表現水準不根據採集樣本的個體的疾病狀態而不同。典型的管家基因包括β-肌動蛋白、甘油醛-3-磷酸脫氫酶和核糖體蛋白P1等。在較佳的實施方案中，對照核酸是已知在處於不同非癌和癌(前)狀態的採集樣本的個體中穩定表現的另一種miRNA。 The difference in performance levels between the obtained target plasma and control plasma can be further normalized to the control nucleic acid, such as the performance level of the housekeeping gene, and the performance level of the housekeeping gene is known not to vary depending on the disease state of the individual collecting the sample. Typical housekeeping genes include β-actin, glyceraldehyde-3-phosphate dehydrogenase, and ribosomal protein P1. In a preferred embodiment, the control nucleic acid is another miRNA that is known to be stably expressed in individuals who are collecting samples in different non-cancer and cancer (pre) states.

但是，代替在任何實驗中確定一個或多個血漿樣本的表現水準，也可以基於實驗證據和/或現有技術資料定義針對特定疾病表型(即疾病狀態)的一個或多個臨界值。在這種情況中，一個或多個靶血漿的相應表現水準可以用一個穩定表現的對照miRNA進行標準化來確定。如果計算的“標準化”的表現水準高於相應定義的臨界值，則說明基因表現上調。反之，如果計算的“標準化”的表現水準低於相應定義的臨界值，則說明microRNA表現下調。 However, instead of determining the performance level of one or more plasma samples in any experiment, one or more threshold values for a particular disease phenotype (ie, disease state) may also be defined based on experimental evidence and/or prior art data. In this case, the corresponding performance level of one or more target plasmas can be determined by normalization with a stable expression of the control miRNA. If the calculated "normalized" performance level is higher than the corresponding defined threshold, then the gene performance is up-regulated. Conversely, if the calculated "normalized" performance level is below the corresponding defined threshold, then the microRNA performance is down-regulated.

在本發明中，術語“鑑定HCC和/或區分其它的HBV感染相關疾病”意為也包括預測和可能性分析(在“診斷”意義上)。本發明公開的組合物和方法意在臨床應用，以決定治療形式，包括治療性干預，診斷標準如疾病階段，和疾病監控和疾病監視。根據本發明，可提供用於檢查受試者狀態的中間結果。這種中間結果可與額外資訊組合以說明醫生、護士或其它從業人員診斷出該物件患有該疾病。或者，本發明可用於通過血漿樣本檢測癌變，並提供有用資訊給醫生以進行診斷。另外，本發明還用於區別HCC和其它HBV感染相關疾病，包括慢性B型肝炎和肝硬化。 In the present invention, the term "identifying HCC and/or distinguishing other diseases associated with HBV infection" is intended to also include prediction and likelihood analysis (in the sense of "diagnosis"). The compositions and methods disclosed herein are intended for clinical use to determine the form of treatment, including therapeutic interventions, diagnostic criteria such as disease stages, and disease surveillance and disease surveillance. In accordance with the present invention, intermediate results for examining the condition of a subject can be provided. This intermediate result can be combined with additional information to indicate that a doctor, nurse, or other practitioner has diagnosed the item as having the condition. Alternatively, the invention can be used to detect cancerous changes through plasma samples and provide useful information to The doctor is going to make a diagnosis. In addition, the invention is also useful for distinguishing between HCC and other HBV infection-related diseases, including chronic hepatitis B and cirrhosis.

在本發明中，所鑑定的一個或多個差異表現的核酸分子一起代表一種標誌物，該標誌物是血漿樣本中HCC的指征。本發明所用的術語“標誌物”是指一組核酸分子(例如miRNA)，其中各個核酸分子的表現水準在HCC患者血漿和對照血漿之間不同。本發明中，所述的標誌物也指一組標誌物並代表最低數目的(不同)核酸分子，每種核酸分子編碼至少一種能鑑定個體的表型狀態的miRNA序列。 In the present invention, one or more of the differentially expressed nucleic acid molecules identified together represent a marker that is indicative of HCC in the plasma sample. The term "marker" as used in the present invention refers to a group of nucleic acid molecules (e.g., miRNAs) in which the performance level of each nucleic acid molecule differs between HCC patient plasma and control plasma. In the present invention, the marker also refers to a group of markers and represents the lowest number of (different) nucleic acid molecules, each nucleic acid molecule encoding at least one miRNA sequence capable of identifying the phenotypic state of the individual.

典型地，包含在HCC診斷標誌物中的核酸分子是人類序列，下文稱為“has”(智人(Homo sapiens))。 Typically, the nucleic acid molecule contained in the HCC diagnostic marker is a human sequence, hereinafter referred to as "has" ( Homo sapiens ).

特佳地，所述的多種核酸分子包含編碼hsa-miR-122(SEQ ID NO：1)、hsa-miR-192(SEQ ID NO：2)、hsa-miR-21(SEQ ID NO：3)、hsa-miR-223(SEQ ID NO：4)、hsa-miR-26a(SEQ ID NO：5)、hsa-miR-27a(SEQ ID NO：6)、hsa-miR-801(SEQ ID NO：7)以及內源性對照hsa-miR-1228(SEQ ID NO：8)的一個或多個核酸分子。 Particularly preferably, said plurality of nucleic acid molecules comprise hsa-miR-122 (SEQ ID NO: 1), hsa-miR-192 (SEQ ID NO: 2), hsa-miR-21 (SEQ ID NO: 3) hsa-miR-223 (SEQ ID NO: 4), hsa-miR-26a (SEQ ID NO: 5), hsa-miR-27a (SEQ ID NO: 6), hsa-miR-801 (SEQ ID NO: 7) and one or more nucleic acid molecules of the endogenous control hsa-miR-1228 (SEQ ID NO: 8).

上面提到的microRNA的核酸序列列在表1中。 The nucleic acid sequences of the above mentioned microRNAs are listed in Table 1.

本發明公開的所有microRNA序列均已經儲存在miRBase資料庫中(http：//microrna.sanger.ac.uk/；也參見Griffiths-Jones S.et al.(2008)Nucl.Acids Res.36,D154-D158)。 All of the microRNA sequences disclosed in the present invention have been stored in the miRBase database (http://microrna.sanger.ac.uk/; see also Griffiths-Jones S. et al. (2008) Nucl. Acids Res. 36, D154 -D158).

編碼hsa-miR-801、hsa-miR-192或hsa-miR-21的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調，編碼hsa-miR-122、hsa-miR-26a、hsa-miR-27a或hsa-miR-223的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調，編碼hsa-miR-1228的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比未改變。 Any one or more nucleic acid molecules encoding hsa-miR-801, hsa-miR-192 or hsa-miR-21 are up-regulated in at least one target plasma compared to the performance in at least one control plasma, encoding hsa Any one or more nucleic acid molecules of -miR-122, hsa-miR-26a, hsa-miR-27a or hsa-miR-223 behaved in at least one target plasma compared to the performance in at least one control plasma Downregulation, the performance of any one or more nucleic acid molecules encoding hsa-miR-1228 in at least one target plasma is unchanged compared to the performance in at least one control plasma.

本發明所用的術語“任何一個或多個核酸分子”或“所述多個核酸分子中的一個或多個”，是指所述的多個核酸分子的任何一個子群組，如任何一個、任何兩個、任何三個、任何四個、任何五個、任何六個、任何七個、任何八個、任何九個、任何十個等等核酸分子，每個核酸分子編碼至少一個microRNA序列。 The term "any one or more nucleic acid molecules" or "one or more of the plurality of nucleic acid molecules" as used in the present invention refers to any one of the plurality of nucleic acid molecules, such as any one, Any two, any three, any four, any five, any six, any seven, any eight Any nine, any ten, etc. nucleic acid molecules, each nucleic acid molecule encoding at least one microRNA sequence.

在最佳的實施方案中，所述的多個核酸分子包含分別編碼hsa-miR-122(SEQ ID NO：1)、hsa-miR-192(SEQ ID NO：2)、hsa-miR-21(SEQ ID NO：3)、hsa-miR-223(SEQ ID NO：4)、hsa-miR-26a(SEQ ID NO：5)、hsa-miR-27a(SEQ ID NO：6)、hsa-miR-801(SEQ ID NO：7)以及hsa-miR-1228(SEQ ID NO：8)的8個核酸分子的組合。所述的8個核酸分子的組合包含於如下邏輯回歸模型：logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209* hsa-miR-801 In a most preferred embodiment, said plurality of nucleic acid molecules comprises hsa-miR-122 (SEQ ID NO: 1), hsa-miR-192 (SEQ ID NO: 2), hsa-miR-21, respectively. SEQ ID NO: 3), hsa-miR-223 (SEQ ID NO: 4), hsa-miR-26a (SEQ ID NO: 5), hsa-miR-27a (SEQ ID NO: 6), hsa-miR- Combination of 8 nucleic acid molecules of 801 (SEQ ID NO: 7) and hsa-miR-1228 (SEQ ID NO: 8). The combination of the 8 nucleic acid molecules is included in the following logistic regression model: logit (p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21- 0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209* hsa-miR-801

其中，hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a以及hsa-miR-801的表現水準是以hsa-miR-1228為內源性對照檢測得到。 Among them, the performance levels of hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR-27a and hsa-miR-801 are hsa- miR-1228 was detected as an endogenous control.

所述的模型中的logit(p=HCC)值在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調，hsa-miR-1228在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比未改變。 The logit (p=HCC) value in the model is up-regulated in at least one target plasma compared to the performance in at least one control plasma, and the performance of hsa-miR-1228 in at least one target plasma is The performance in at least one control plasma was unchanged.

本發明所用的術語“核酸分子組合”是指將至少兩個核酸表現水準作為一個整體來使用。較佳將相對變化或通過一個公式計算出的結果作為一個整體來使用。 The term "nucleic acid molecule combination" as used in the present invention refers to the use of at least two nucleic acid expression levels as a whole. It is preferable to use the relative change or the result calculated by a formula as a whole.

在第二個方面，本發明公開了一種肝癌診斷套組，其包含一種HCC診斷標誌物，所述的HCC診斷標誌物，由多種核酸分子組成，每種核酸分子編碼至少一個microRNA 序列。 In a second aspect, the present invention discloses a liver cancer diagnostic kit comprising an HCC diagnostic marker, the HCC diagnostic marker comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding at least one microRNA sequence.

在最佳的實施方案中，所述的多個核酸分子包含分別編碼hsa-miR-122(SEQ ID NO：1)、hsa-miR-192(SEQ ID NO：2)、hsa-miR-21(SEQ ID NO：3)、hsa-miR-223(SEQ ID NO：4)、hsa-miR-26a(SEQ ID NO：5)、hsa-miR-27a(SEQ ID NO：6)、hsa-miR-801(SEQ ID NO：7)以及hsa-miR-1228(SEQ ID NO：8)的8個核酸分子的組合。本發明的套組還包含含有上述8個核酸分子的組合的邏輯回歸模型：logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209* hsa-miR-801 In a most preferred embodiment, said plurality of nucleic acid molecules comprises hsa-miR-122 (SEQ ID NO: 1), hsa-miR-192 (SEQ ID NO: 2), hsa-miR-21, respectively. SEQ ID NO: 3), hsa-miR-223 (SEQ ID NO: 4), hsa-miR-26a (SEQ ID NO: 5), hsa-miR-27a (SEQ ID NO: 6), hsa-miR- Combination of 8 nucleic acid molecules of 801 (SEQ ID NO: 7) and hsa-miR-1228 (SEQ ID NO: 8). The kit of the present invention further comprises a logistic regression model comprising a combination of the above 8 nucleic acid molecules: logit (p=HCC) = -1.442 - 0.292 * hsa - miR - 12 + 0.4511 * hsa - miR - 19 + 0.6112 * hsa - miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209* hsa-miR-801

在第三方面，本發明公開了一種用於區分至少一個HCC患者的血漿和至少一個健康人的血漿的套組，其包含一種HCC診斷標誌物，所述的HCC診斷標誌物，由多種核酸分子組成，每種核酸分子編碼至少一個microRNA序列，所述的多種核酸分子包含至少一種編碼microRNA序列的核酸分子，其在至少一種靶血漿中與在至少一種對照血漿中差異表現，所述的至少一種對照血漿來自健康人。 In a third aspect, the present invention discloses a kit for distinguishing plasma of at least one HCC patient from plasma of at least one healthy person, comprising an HCC diagnostic marker, said HCC diagnostic marker, comprising a plurality of nucleic acid molecules Composition, each nucleic acid molecule encoding at least one microRNA sequence comprising at least one nucleic acid molecule encoding a microRNA sequence that differs in at least one target plasma and in at least one control plasma, said at least one Control plasma is from healthy people.

編碼hsa-miR-122、hsa-miR-801、hsa-miR-192或hsa-miR-21的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調，編碼hsa-miR-26a、hsa-miR-27a或hsa-miR-223的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調，編碼hsa-miR-1228的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比未改變。 Expression of any one or more nucleic acid molecules encoding hsa-miR-122, hsa-miR-801, hsa-miR-192 or hsa-miR-21 in at least one target plasma and in at least one control plasma Ratio is up-regulated, encoding any of hsa-miR-26a, hsa-miR-27a or hsa-miR-223 or The performance of a plurality of nucleic acid molecules in at least one target plasma is down-regulated compared to the performance in at least one control plasma, and the performance of any one or more nucleic acid molecules encoding hsa-miR-1228 in at least one target plasma The performance in at least one control plasma was unchanged.

在第四方面，本發明公開了一種用於區分至少一個HCC患者的血漿和至少一個慢性B型肝炎的血漿的套組，其包含一種HCC診斷標誌物，所述的HCC診斷標誌物，由多種核酸分子組成，每種核酸分子編碼至少一個 microRNA序列，所述的多種核酸分子包含至少一種編碼microRNA序列的核酸分子，其在至少一種靶血漿中與在至少一種對照血漿中差異表現，所述的至少一種對照血漿來自慢性B型肝炎患者。 In a fourth aspect, the present invention discloses a kit for distinguishing plasma of at least one HCC patient from at least one chronic hepatitis B virus, comprising an HCC diagnostic marker, said HCC diagnostic marker, a nucleic acid molecule composition, each nucleic acid molecule encoding at least one A microRNA sequence comprising at least one nucleic acid molecule encoding a microRNA sequence that is differentially expressed in at least one target plasma and in at least one control plasma, said at least one control plasma being from a chronic hepatitis B patient.

編碼hsa-miR-801或hsa-miR-21的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調，編碼hsa-miR-122、hsa-miR-192、hsa-miR-26a、hsa-miR-27a或hsa-miR-223的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調，編碼hsa-miR-1228的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比未改變。 Any one or more nucleic acid molecules encoding hsa-miR-801 or hsa-miR-21 are up-regulated in at least one target plasma compared to expression in at least one control plasma, encoding hsa-miR-122, hsa Any one or more nucleic acid molecules of -miR-192, hsa-miR-26a, hsa-miR-27a or hsa-miR-223 behaved in at least one target plasma compared to the performance in at least one control plasma Downregulation, the performance of any one or more nucleic acid molecules encoding hsa-miR-1228 in at least one target plasma is unchanged compared to the performance in at least one control plasma.

在最佳的實施方案中，所述的多個核酸分子包含分別編碼hsa-miR-122(SEQ ID NO：1)、hsa-miR-192(SEQ ID NO：2)、hsa-miR-21(SEQ ID NO：3)、hsa-miR-223(SEQ ID NO：4)、hsa-miR-26a(SEQ ID NO：5)、hsa-miR-27a(SEQ ID NO：6)、hsa-miR-801(SEQ ID NO：7)以及hsa-miR-1228(SEQ ID NO：8)的8個核酸分子的組合。本發明的套組還包含含有上述8個核酸分子的組合的邏輯回歸模型： logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209* hsa-miR-801 In a most preferred embodiment, said plurality of nucleic acid molecules comprises hsa-miR-122 (SEQ ID NO: 1), hsa-miR-192 (SEQ ID NO: 2), hsa-miR-21, respectively. SEQ ID NO: 3), hsa-miR-223 (SEQ ID NO: 4), hsa-miR-26a (SEQ ID NO: 5), hsa-miR-27a (SEQ ID NO: 6), hsa-miR- Combination of 8 nucleic acid molecules of 801 (SEQ ID NO: 7) and hsa-miR-1228 (SEQ ID NO: 8). The kit of the invention further comprises a logistic regression model comprising a combination of the above 8 nucleic acid molecules: Logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a- 0.3542*hsa-miR-27a+0.209* hsa-miR-801

在第五方面，本發明公開了一種用於區分至少一個HCC患者的血漿和至少一個肝硬化患者的血漿的套組，其包含一種HCC診斷標誌物，所述的HCC診斷標誌物，由多種核酸分子組成，每種核酸分子編碼至少一個microRNA序列，所述的多種核酸分子包含至少一種編碼microRNA序列的核酸分子，其在至少一種靶血漿中與在至少一種對照血漿中差異表現，所述的至少一種對照血漿來自肝硬化患者。 In a fifth aspect, the present invention discloses a kit for distinguishing plasma of at least one HCC patient from plasma of at least one cirrhotic patient, comprising an HCC diagnostic marker, said HCC diagnostic marker, comprising a plurality of nucleic acids a molecular composition, each nucleic acid molecule encoding at least one microRNA sequence, the plurality of nucleic acid molecules comprising at least one nucleic acid molecule encoding a microRNA sequence that differs in at least one target plasma and in at least one control plasma, said at least One control plasma is from a patient with cirrhosis.

編碼hsa-miR-801的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調，編碼hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-26a、hsa-miR-27a或hsa-miR-223的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調，編碼hsa-miR-1228的任何一個或多個核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比未改變。 Any one or more nucleic acid molecules encoding hsa-miR-801 are up-regulated in at least one target plasma compared to expression in at least one control plasma, encoding hsa-miR-122, hsa-miR-192, hsa Any one or more nucleic acid molecules of -miR-21, hsa-miR-26a, hsa-miR-27a or hsa-miR-223 behaved in at least one target plasma compared to the performance in at least one control plasma Downregulation, the performance of any one or more nucleic acid molecules encoding hsa-miR-1228 in at least one target plasma is unchanged compared to the performance in at least one control plasma.

在最較佳的實施方案中，所述的多個核酸分子包含分別編碼hsa-miR-122(SEQ ID NO：1)、hsa-miR-192(SEQ ID NO：2)、hsa-miR-21(SEQ ID NO：3)、hsa-miR-223(SEQ ID NO：4)、hsa-miR-26a(SEQ ID NO：5)、hsa-miR-27a(SEQ ID NO：6)、hsa-miR-801(SEQ ID NO：7)以及hsa-miR-1228(SEQ ID NO：8)的8個核酸分子的組合。本發明的套組還包含含有上述8個核酸分子的組合的邏輯回歸模型：logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209* hsa-miR-801 In a most preferred embodiment, said plurality of nucleic acid molecules comprises hsa-miR-122 (SEQ ID NO: 1), hsa-miR-192 (SEQ ID NO: 2), hsa-miR-21, respectively. (SEQ ID NO: 3), hsa-miR-223 (SEQ ID NO: 4), hsa-miR-26a (SEQ ID NO: 5), hsa-miR-27a (SEQ ID NO: 6), hsa-miR a combination of -801 (SEQ ID NO: 7) and 8 nucleic acid molecules of hsa-miR-1228 (SEQ ID NO: 8). The kit of the present invention further comprises a logistic regression model comprising a combination of the above 8 nucleic acid molecules: logit (p=HCC) = -1.442 - 0.292 * hsa - miR - 12 + 0.4511 * hsa - miR - 19 + 0.6112 * hsa - miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209* hsa-miR-801

在第六方面，本發明公開了一種確定肝癌診斷標誌物的方法，包括：(a)在一個或多個靶血漿中確定多個核酸分子的表現水準，每個核酸分子編碼至少一個microRNA序列；(b)在一個或多個對照血漿中確定上述多個核酸分子的表現水準；(c)通過對比在步驟(a)和(b)中獲得的多個核酸分子的相應表現水準，來從所述多個核酸分子中鑑定出在所述的靶血漿和所述的對照血漿中差異表現的一個或多個核酸分子，將在所述的靶血漿和所述的對照血漿中差異表現的一個或多個核酸分子作為肝癌診斷標誌物，來鑑定一個或更多個肝癌患者的靶血漿。 In a sixth aspect, the present invention discloses a method for determining a liver cancer diagnostic marker, comprising: (a) determining a performance level of a plurality of nucleic acid molecules in one or more target plasmas, each nucleic acid molecule encoding at least one microRNA sequence; (b) determining the performance level of said plurality of nucleic acid molecules in one or more control plasmas; (c) by comparing the respective performance levels of the plurality of nucleic acid molecules obtained in steps (a) and (b) One or more nucleic acid molecules which are differentially expressed in the target plasma and the control plasma, and one or more of the differential expressions in the target plasma and the control plasma are identified in the plurality of nucleic acid molecules A plurality of nucleic acid molecules are used as diagnostic markers for liver cancer to identify target plasma of one or more liver cancer patients.

本發明通過附圖和如下實施例進一步描述，所述的附圖和實施例只是為了例證本發明的特定實施方案，不應理解為以任何方式限制本發明範圍之意。 The invention is further described by the accompanying drawings and the accompanying drawings, which are to be construed as illustrative only.

實施例1：血漿樣本採集和製備： Example 1: Plasma sample collection and preparation:

本發明中的試驗已得到本地倫理委員會的批准並獲得所有患者的知情同意。本發明中microRNA生物標誌物的篩選階段、訓練階段以及驗證階段的實驗設計如圖1所示。用被推薦的血漿microRNA組合來鑑別HCC患者的血漿樣本的主要方法步驟如圖2所示。 The trials in the present invention have been approved by the local ethics committee and informed consent has been obtained from all patients. The experimental design of the screening phase, the training phase, and the verification phase of the microRNA biomarkers in the present invention is shown in FIG. The main method steps for identifying plasma samples from HCC patients using the recommended combination of plasma microRNAs are shown in Figure 2.

在2008年8月到2010年6月間，934個滿足合格標準(圖2)的血液樣本被預先從上海中山醫院和公共衛生中心採集。這些樣本從167個健康捐獻者(健康組)，169個慢性B型肝炎患者(CHB組)，141個HBV感染後肝硬化患者(肝硬化組)，以及457個HBV感染相關HCC患者(HCC組)獲得。這些樣本被按照時間先後順序劃歸為3個階段(圖1)。患者的臨床表現被總結在表3和4中。 Between August 2008 and June 2010, 934 blood samples meeting the eligibility criteria (Figure 2) were pre-collected from Shanghai Zhongshan Hospital and Public Health Center. These samples ranged from 167 healthy donors (healthy group), 169 chronic hepatitis B patients (CHB group), 141 HBV-infected patients with cirrhosis (cirrhosis group), and 457 HBV-infected HCC patients (HCC group). )obtain. These samples were classified into three phases in chronological order (Figure 1). The clinical presentation of the patients is summarized in Tables 3 and 4.

抽取外周血(4 ml)放入EDTA管中，30分鐘內，將EDTA管在820g條件下離心10分鐘。然後，將1ml血漿轉移到1.5ml管中，在16，000g條件下離心10分鐘以除去殘留的細胞碎屑。隨後將上清液轉移到乾淨的管中，在-80℃儲存。 Peripheral blood (4 ml) was taken and placed in an EDTA tube, and the EDTA tube was centrifuged at 820 g for 10 minutes in 30 minutes. Then, 1 ml of plasma was transferred to a 1.5 ml tube and centrifuged at 16,000 g for 10 minutes to remove residual cell debris. The supernatant was then transferred to a clean tube and stored at -80 °C.

用mirVana PARIS microRNA分離套組(mirVana PARIS miRNA Isolation kit)按照廠家(Ambion，Austin，TX)提供的使用說明來抽提總RNA，用NanoDrop 1000分光光度計(NanoDrop Technologies，Waltham，MA)來定量檢測其濃度。 Total RNA was extracted using the mirVana PARIS miRNA Isolation kit according to the instructions provided by the manufacturer (Ambion, Austin, TX) and quantified using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Waltham, MA). Its concentration.

實施例2：microRNA微陣列分析： Example 2: MicroRNA microarray analysis:

用Agilent microRNA微陣列平臺(Agilent microRNA microarray platform，Agilent Technologies，Santa Clara，CA，USA)對特定血漿樣本中的microRNA進行定性分析。該微陣列包含來自Sanger database v.10.1的723個人類microRNA的探針。將來自137個血漿樣本中任一個的總RNA(100ng)摻入單色CY3用於標記。用XDR掃描器(PMT100，PMT5)對基因晶片進行掃描，按照Agilent microRNA微陣列系統的規程進行標記和雜交。通過Feature Extraction Software Rev.9.5.3(Agilent Technologies，Santa Clara，CA)將微陣列圖像資訊轉化成光點強度值。將除掉背景後的信號用穩定的內源性對照hsa-miR-1228進行標準化。然後，進行底數為2的log轉換。一個陳列上重複點的片間變異係數(CV)超過15%或可檢測到的信號小於5%為不可信樣本，排除上述不可信樣本後，進行進一步分析。 Qualitative analysis of microRNAs in specific plasma samples was performed using an Agilent microRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA). The microarray contained probes from the 723 human microRNA of Sanger database v.10.1. Total RNA (100 ng) from any of 137 plasma samples was spiked into monochromatic CY3 for labeling. The gene wafers were scanned using an XDR scanner (PMT100, PMT5) and labeled and hybridized according to the protocol of the Agilent microRNA microarray system. Microarray image information was converted to spot intensity values by Feature Extraction Software Rev. 9.5.3 (Agilent Technologies, Santa Clara, CA). The signal after background removal was normalized with the stable endogenous control hsa-miR-1228. Then, a log conversion with a base of 2 is performed. An inter-chip coefficient of variation (CV) of more than 15% on a display or a detectable signal of less than 5% is an untrustworthy sample, and further analysis is performed after excluding the above untrusted sample.

受試者的人口學特徵通過描述性統計來報告。卡方或T檢驗被用於訓練集和驗證集之間的比較(表5)。Kruskal-Wallis檢驗被用於HCC組、健康組、CHB組以及肝硬化組之間的總體比較。Mann-Whitney未配對檢驗被用於兩組之間的比較。這些檢驗得到的p值全部通過Benjamini-Hochberg方法進行多重檢驗校正。所有的p值都是雙側的。 The demographic characteristics of the subjects are reported by descriptive statistics. A chi-square or T-test is used for comparison between the training set and the validation set (Table 5). The Kruskal-Wallis test was used for the HCC group, the health group, the CHB group, and Overall comparison between the cirrhotic groups. The Mann-Whitney unpaired test was used for comparison between the two groups. The p values obtained from these tests were all corrected by the multiple test by the Benjamini-Hochberg method. All p values are bilateral.

採用以下標準來選擇在微陣列上有可檢出信號的候選microRNA用於驗證：(1)HCC組、健康組、CHB組以及肝硬化組之間的Kruskal-Wallis檢驗的校正後的p值<0.001；(2)HCC組和健康組之間的Mann-Whitney未配對檢驗的校正後的p值<0.05；(3)HCC組和CHB組之間的Mann-Whitney未配對檢驗的校正後的p值<0.00000001；以及(4)HCC組和肝硬化組之間的Mann-Whitney未配對檢驗的校正後的p值<0.0001。 Candidate microRNAs with detectable signals on the microarray were selected for validation using the following criteria: (1) The corrected p-value of the Kruskal-Wallis test between the HCC, healthy, CHB, and cirrhotic groups < 0.001; (2) The adjusted p-value of the Mann-Whitney unpaired test between the HCC group and the healthy group was <0.05; (3) the corrected p of the Mann-Whitney unpaired test between the HCC group and the CHB group. Values <0.00000001; and (4) The corrected p-value of the Mann-Whitney unpaired test between the HCC group and the cirrhosis group was <0.0001.

實施例3：137個樣本的微陣列資料 Example 3: 137 samples of microarray data

用於進一步驗證的15個候選microRNA在微陣列分析中的表現如表5所示，滿足選擇標準的microRNA用黑體表示。 The performance of 15 candidate microRNAs for further validation in microarray analysis is shown in Table 5, and microRNAs that meet the selection criteria are indicated in bold.

實施例4：通過102個血漿樣本評價候選microRNA Example 4: Evaluation of candidate microRNAs by 102 plasma samples

採用102個血漿樣本的獨立群組以及不同的技術平臺來對微陣列篩選出的15個候選microRNA進行評價。使用TaqMan MicroRNA檢測套組(TaqMan MicroRNA assay kit，Applied Biosystems，Foster City，CA，USA)按照廠家的使用說明操作。所有的檢測都重複3次。以hsa-miR-1228的表現水準作為內源性對照。在超過102個樣本的20%中表現出CT值大於35個循環的microRNA被排除，不再進行進一步分析。 Fifteen candidate microRNAs screened by the microarray were evaluated using a separate set of 102 plasma samples and different technology platforms. The TaqMan MicroRNA assay kit (TaqMan MicroRNA assay kit, Applied Biosystems, Foster City, CA, USA) was used according to the manufacturer's instructions for use. All tests were repeated 3 times. The performance level of hsa-miR-1228 was used as an endogenous control. MicroRNAs showing CT values greater than 35 cycles in 20% of more than 102 samples were excluded and no further analysis was performed.

Kruskal-Wallis檢驗被用於HCC組、健康組、CHB組以及肝硬化組之間的總體比較。Mann-Whitney檢驗被用於兩組之間的比較。這些檢驗得到的p值全部通過Benjamini-Hochberg方法進行多重檢驗校正。所有的p值都是雙側的。 The Kruskal-Wallis test was used for overall comparisons between the HCC group, the healthy group, the CHB group, and the cirrhosis group. The Mann-Whitney test was used for comparison between the two groups. The p values obtained from these tests were all corrected by the multiple test by the Benjamini-Hochberg method. All p values are bilateral.

15個候選microRNA在微陣列分析中的表現如表5所示。15個候選microRNA中的12個通過了品質控制過程。它們中的7個(hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a和hsa-miR-801)在HCC組和對照組之間表現水準具有顯著差異。15個候選microRNA在定量RT-PCR上的表現譜如表6所示。特佳的microRNA(SEQ ID NO：1 to SEQ ID NO：7)用黑體表示。 The performance of 15 candidate microRNAs in microarray analysis is shown in Table 5. Twelve of the 15 candidate microRNAs passed the quality control process. 7 of them (hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR-27a and hsa-miR-801) in HCC There was a significant difference in performance levels between the group and the control group. The performance profiles of 15 candidate microRNAs on quantitative RT-PCR are shown in Table 6. Particularly preferred microRNAs (SEQ ID NO: 1 to SEQ ID NO: 7) are indicated in bold.

對照組包括健康個體、CHB患者以及肝硬化患者。在HCC組和對照組中差異表現的microRNA用黑體表示。ND表示不能確定，因為該microRNA未通過品質控制過程。 The control group included healthy individuals, CHB patients, and patients with cirrhosis. MicroRNAs that differed in the HCC group and the control group were indicated in bold. ND indicates that it cannot be determined because the microRNA did not pass the quality control process.

實施例5：在含有407個樣本的訓練集中建立一個microRNA診斷模型 Example 5: Establish a microRNA diagnostic model in a training set containing 407 samples

在另外的305個血漿樣本上使用定量RT-PCR檢測方法來進一步評價7個差異表現的microRNA的表現譜。 Quantitative RT-PCR assays were used on an additional 305 plasma samples to further evaluate the performance profiles of the seven differentially expressed microRNAs.

407個血漿樣本被結合起來用作訓練集來建立HCC診斷microRNA組合。同樣地，Kruskal-Wallis檢驗被用於HCC 組、健康組、CHB組以及肝硬化組之間的總體比較。Mann-Whitney未配對檢驗被用於兩組之間的比較。這些檢驗得到的p值全部通過Benjamini-Hochberg方法進行多重檢驗校正。所有的p值都是雙側的。 407 plasma samples were combined for use as a training set to establish an HCC diagnostic microRNA combination. Similarly, the Kruskal-Wallis test is used for HCC Overall comparison between group, healthy group, CHB group, and cirrhosis group. The Mann-Whitney unpaired test was used for comparison between the two groups. The p values obtained from these tests were all corrected by the multiple test by the Benjamini-Hochberg method. All p values are bilateral.

逐步邏輯回歸模型被用於基於訓練集選擇microRNA診斷標誌物。被診斷患有HCC的預測概率被用作替代標誌物來建立受試者工作特徵(ROC)曲線。ROC曲線下面積(AUC)被用於評價血清AFP和microRNA組合的診斷效能。MedCalc(10.4.7.0)軟體被用於進行ROC和回歸分析。 A stepwise logistic regression model was used to select microRNA diagnostic markers based on the training set. The predicted probability of being diagnosed with HCC is used as a surrogate marker to establish a receiver operating characteristic (ROC) curve. The area under the ROC curve (AUC) was used to evaluate the diagnostic efficacy of serum AFP and microRNA combinations. The MedCalc (10.4.7.0) software was used for ROC and regression analysis.

7個候選microRNA在訓練集的表現譜和診斷效能如表6所示。microRNA組合的AUC顯著地大於AFP的AUC(0.86 vs.0.76，p<0.001，圖3A)。 The performance profiles and diagnostic performance of the seven candidate microRNAs in the training set are shown in Table 6. The AUC of the microRNA combination was significantly greater than the AUC of AFP (0.86 vs. 0.76, p < 0.001, Figure 3A).

實施例6：在390個樣本的驗證集中驗證上述microRNA組合 Example 6: Verification of the above microRNA combination in a validation set of 390 samples

從訓練集估計的參數被用於獨立的驗證集(390血漿樣本)上預測被診斷患有HCC的概率。同樣地，採用定量RT-PCR檢測方法來進行實驗。預測概率被用來建立受試者工作特徵曲線。 The parameters estimated from the training set were used on an independent validation set (390 plasma samples) to predict the probability of being diagnosed with HCC. Similarly, experiments were performed using quantitative RT-PCR detection methods. The predicted probability is used to establish a receiver operating characteristic curve.

microRNA組合與AFP之間的AUC在驗證集中的比較表明microRNA組合的診斷準確度顯著高於AFP(AUC：0.89 vs.0.68，p<0.001，圖3B)。 A comparison of the AUC between the microRNA combination and AFP in the validation set indicated that the diagnostic accuracy of the microRNA combination was significantly higher than AFP (AUC: 0.89 vs. 0.68, p < 0.001, Figure 3B).

microRNA組合與AFP區分HCC組和健康組、CHB組以及肝硬化組的效能也進行了對比(圖4)。該分析證明了microRNA組合與AFP皆可區分HCC組和非HCC。然而，microRNA組合的AUC顯著大於AFP(HCC與健康組：0.95 vs.0.64，p<0.001，HCC與CHB：0.85 vs.0.62，p<0.001 and HCC與肝硬化組：0.89 vs.0.78，p=0.002)。 The efficacy of microRNA combination and AFP distinguishing between HCC and healthy, CHB, and cirrhosis groups was also compared (Figure 4). This analysis demonstrates that both microRNA combinations and AFPs distinguish between HCC and non-HCC. However, the AUC of the microRNA combination was significantly greater than that of the AFP (HCC vs. healthy group: 0.95 vs. 0.64, p<0.001, HCC and CHB: 0.85 vs. 0.62, p<0.001 and HCC vs. cirrhosis: 0.89 vs. 0.78, p= 0.002).

實施例7：microRNA組合與AFP在不同BCLC分期的診斷表現 Example 7: Diagnostic performance of microRNA combination and AFP in different BCLC stages

microRNA組合與AFP在不同BCLC分期的診斷表現被進一步評價(圖5)。在診斷早期和中期HCC(BCLC 0、A和B期)方面，microRNA組合的診斷表現顯著高於AFP(BCLC 0期，AUC 0.94 vs.0.68，p<0.001；BCLC A期，AUC 0.90 vs.0.65，p<0.001；BCLC B期，AUC 0.85 vs.0.74，p<0.001；圖5A，5B and 5 C)。當microRNA組合和AFP之間的比較聚焦在BCLC C期患者上時，其診斷表現並無顯著差異(AUC 0.80 vs.0.79，p=0.24，圖5D)。 The diagnostic performance of microRNA combination and AFP at different BCLC stages was further evaluated (Fig. 5). In the diagnosis of early and intermediate HCC (BCLC 0, A and B), the diagnostic performance of microRNA combination was significantly higher than AFP (BCLC stage 0, AUC 0.94 vs. 0.68, p < 0.001; BCLC stage A, AUC 0.90 vs. 0.65) , p < 0.001; BCLC B phase, AUC 0.85 vs. 0.74, p < 0.001; Figures 5A, 5B and 5 C). When the comparison between microRNA combination and AFP was focused on BCLC stage C patients, there was no significant difference in diagnostic performance (AUC 0.80 vs. 0.79, p=0.24, Figure 5D).

實施例8：microRNA組合在AFP正常(20ng/ml)組和高AFP(>20ng/ml)組中的診斷表現 Example 8: MicroRNA combination is normal in AFP ( Diagnostic performance in the 20 ng/ml group and the high AFP (>20 ng/ml) group

按照AFP水準評估microRNA組合診斷準確度。在AFP正常(20ng/ml)組中，microRNA組合的AUC顯著大於AFP的AUC(0.87 vs.0.63，p<0.001，圖6A)。在高AFP(>20ng/ml)組中，microRNA組合的AUC仍然顯著大於AFP的AUC(0.90 vs.0.69，p<0.001，圖6B)。 The accuracy of microRNA combination diagnosis is assessed according to AFP levels. Normal in AFP ( In the 20 ng/ml) group, the AUC of the microRNA combination was significantly greater than the AUC of AFP (0.87 vs. 0.63, p < 0.001, Figure 6A). In the high AFP (>20 ng/ml) group, the AUC of the microRNA combination was still significantly greater than the AUC of AFP (0.90 vs. 0.69, p < 0.001, Figure 6B).

實施例9：HCC切除術前後的microRNA表現變化 Example 9: MicroRNA expression changes before and after HCC resection

分別在術前和術後第6天，取接受肝外科切除術的54個HCC患者的血液樣本。使用定量RT-PCR來監控microRNA表現譜的變化。所有的檢測都重複3次，以減少誤差。hsa-miR-1228的表現水準用作內源性對照。 Blood samples from 54 HCC patients undergoing hepatectomy were performed before surgery and on the 6th day after surgery. Quantitative RT-PCR was used to monitor changes in microRNA expression profiles. All tests were repeated 3 times to reduce errors. The performance level of hsa-miR-1228 was used as an endogenous control.

觀察得到，hsa-miR-21、hsa-miR-192和AFP的表現水準在HCC切除術後顯著降低(平均差異分別為0.77、1.74和6.66，p值分別為p<0.001、p=0.03和p<0.001，圖7)。hsa-miR-223，hsa-miR-26a和hsa-miR-27a的術後表現水準與術前表現水準相比有所升高(平均差異分別為-0.61、-0.81和-0.55，p值分別為p=0.02、0.06和0.06)。hsa-miR-122和hsa-miR-801的表現水準並未由於手術產生顯著變化。 It was observed that the performance levels of hsa-miR-21, hsa-miR-192 and AFP were significantly reduced after HCC resection (mean differences were 0.77, 1.74 and 6.66, respectively, p values were p<0.001, p=0.03 and p, respectively). <0.001, Figure 7). The postoperative performance level of hsa-miR-223, hsa-miR-26a and hsa-miR-27a was increased compared with the preoperative performance level (mean differences were -0.61, -0.81 and -0.55, respectively, p values were respectively For p = 0.02, 0.06 and 0.06). The performance levels of hsa-miR-122 and hsa-miR-801 were not significantly altered by surgery.

所獲得的結果證實microRNA在HCC患者血漿中的特異性表現。因此，這裡規定的microRNA集可作為獨特的microRNA生物標誌物，通過HCC血漿microRNA表現譜分析，不僅可以早期診斷肝癌的發生，也可區別HCC和慢性B型肝炎以及肝硬化。 The results obtained confirm the specific expression of microRNAs in the plasma of HCC patients. Therefore, the microRNA set specified here can be used as a unique microRNA biomarker. Through HCC plasma microRNA expression profiling, it can not only diagnose early liver cancer, but also distinguish between HCC and chronic hepatitis B and cirrhosis.

本發明對於血漿microRNA表現生物標誌物的鑑別和驗證提供了可在血液樣本中進行篩選、早期診斷、區別診斷HCC的獨特的分子標誌物。 The present invention provides a unique molecular marker for screening, early diagnosis, and differential diagnosis of HCC in blood samples for the identification and verification of biomicromarker biomarkers.

在此舉例描述的本發明可以適當地在不存在非本文特別揭示的任何要素、限制的條件下實施。因此，例如術語“包含”、“包括”“含有”等應解讀為具有廣泛含義且無限制。另外，本文應用的術語和表現用於描述而並非限制本發明，且沒有使用這些術語和表現排除所表現或描述的特徵的任何等價形式或其組成部分之意，但是應意識到在本發明請求保護的範圍內可以進行各種修改。因此，應理解儘管本發明通過即時方式和選擇性特徵進行特別揭示，本領域技術人員可以得出對本發明的修改和改變，這種修改和改變應被認為在本發明的範圍之內。 The invention as exemplified herein may be suitably carried out in the absence of any elements or limitations not specifically disclosed herein. Thus, for example, the terms "comprises", "comprising", "comprising", etc. In addition, the terms and expressions of the present invention are used to describe and not to limit the invention, and the use of these terms and expressions to exclude any equivalent forms of the features that are expressed or described, or the components thereof, is intended to be Various modifications can be made within the scope of requesting protection. Therefore, it is to be understood that modifications and variations of the present invention may be made without departing from the scope of the invention.

本發明進行了一般地並上位地描述。落入上位描述範圍內的每個較窄的下位概念和次上位概念也組成了本發明的一部分。包括帶有從其中除去任何主題的條件或否定限制的上位描述，而無論所除去的主題是否在本發明中特別引述。 The invention has been described generally and generically. Each of the narrower subordinate concepts and subordinate superior concepts falling within the scope of the above description also form part of the present invention. The above description includes the above description of the condition or the negative limitation of any subject matter, regardless of whether the removed subject matter is specifically recited in the present invention.

其它實施方式在申請專利範圍內。另外，當本發明的各個特徵或方面用馬庫斯方式描述時，本領域技術人員能意識到本發明也以馬庫斯的任何成員或成員子群組方式被描述。 Other embodiments are within the scope of the patent application. In addition, when various features or aspects of the invention are described in the Marcus manner, those skilled in the art will appreciate that the invention is also described in the form of any member or subgroup of members of Marcus.

圖1為本發明用於鑑定至少一個HCC靶血漿，特別是存在早期HCC(BCLC 0期和A期)的microRNA組合的篩選、訓練以及驗證階段的實驗設計流程圖。 1 is a flow chart showing the experimental design of the screening, training, and validation stages of the microRNA combination for identifying at least one HCC target plasma, particularly in the presence of early HCC (BCLC Phase 0 and Phase A).

圖2為確定本發明用於診斷HCC，特別是診斷早期HCC(BCLC 0期和A期)患者的血液中microRNA組合的主要方法步驟圖。對照組包括健康、慢性B型肝炎和肝硬化受試者。 Figure 2 is a diagram showing the main method steps for determining the microRNA combination in the blood of the present invention for diagnosing HCC, particularly for diagnosing early HCC (BCLC Stage 0 and Phase A) patients. The control group included healthy, chronic hepatitis B and cirrhosis Subject.

圖3說明瞭包含本發明用於確定至少一個HCC靶血漿的較佳microRNA組合(hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a和hsa-miR-801)的邏輯回歸模型。A)訓練集(n=407)中HCC組與對照組比較時microRNA組合的診斷價值的ROC曲線圖。與AFP(AUC=0.76)相比，microRNA組合在區分HCC組血漿和對照組血漿時具有顯著高的診斷準確度(AUC=0.86)。B)驗證集(n=390)中HCC組與對照組比較時microRNA組合的診斷價值的ROC曲線圖。與AFP(AUC=0.68)相比，microRNA組合在區分HCC組血漿和對照組血漿時具有顯著高的診斷準確度(AUC=0.89)。 Figure 3 illustrates a preferred microRNA combination comprising the invention for determining at least one HCC target plasma (hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a) Logistic regression models for hsa-miR-27a and hsa-miR-801). A) ROC plot of the diagnostic value of the microRNA combination when the HCC group was compared to the control group in the training set (n=407). Compared to AFP (AUC = 0.76), the microRNA combination had a significantly higher diagnostic accuracy (AUC = 0.86) in distinguishing between HCC group plasma and control group plasma. B) ROC plot of the diagnostic value of the microRNA combination when the HCC group was compared to the control group in the validation set (n=390). Compared to AFP (AUC = 0.68), the microRNA combination had a significantly higher diagnostic accuracy (AUC = 0.89) in distinguishing between HCC group plasma and control group plasma.

圖4說明瞭包含本發明用於進一步區分HCC患者血漿和健康個體、慢性B型肝炎患者或肝硬化患者的血漿的較佳microRNA組合(hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a和hsa-miR-801)的邏輯回歸模型。A)驗證集(n=390)中HCC組與健康組比較時microRNA組合的診斷價值的ROC曲線圖。與AFP(AUC=0.64)相比，microRNA組合在區分HCC患者血漿和健康個體血漿時具有顯著高的診斷準確度(AUC=0.95)。B)驗證集(n=390)中HCC組與慢性B型肝炎組比較時microRNA組合的診斷價值的ROC曲線圖。與AFP(AUC=0.62)相比，microRNA組合在區分HCC患者血漿和慢性B型肝炎患者的血漿時具有顯著高的診斷準確度(AUC=0.85)。C)驗證集(n=390)中HCC組與肝硬化組比較時microRNA組合的診斷價值的ROC曲線圖。與AFP (AUC=0.78)相比，microRNA組合在區分HCC患者血漿和肝硬化患者的血漿時具有顯著高的診斷準確度(AUC=0.89)。 Figure 4 illustrates a preferred microRNA combination (hsa-miR-122, hsa-miR-192, hsa-miR) comprising the present invention for further distinguishing plasma from HCC patients with plasma and healthy individuals, chronic hepatitis B patients or cirrhotic patients. -21, Logistic regression model of hsa-miR-223, hsa-miR-26a, hsa-miR-27a and hsa-miR-801). A) ROC plot of the diagnostic value of the microRNA combination when the HCC group was compared to the healthy group in the validation set (n=390). Compared to AFP (AUC = 0.64), the microRNA combination has a significantly higher diagnostic accuracy (AUC = 0.95) in distinguishing between HCC patient plasma and healthy individual plasma. B) ROC plot of the diagnostic value of microRNA combination in the HCC group versus the chronic hepatitis B group in the validation set (n=390). Compared to AFP (AUC = 0.62), the microRNA combination has a significantly higher diagnostic accuracy (AUC = 0.85) in distinguishing plasma from HCC patient plasma and chronic hepatitis B patients. C) ROC plot of the diagnostic value of the microRNA combination when the HCC group was compared to the cirrhosis group in the validation set (n=390). With AFP (AUC = 0.78) The microRNA combination has a significantly higher diagnostic accuracy (AUC = 0.89) in distinguishing plasma from patients with HCC patients' plasma and cirrhosis.

圖5說明瞭包含本發明用於鑑定不同BCLC分期的HCC的至少一個靶血漿的較佳microRNA組合(hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a和hsa-miR-801)的邏輯回歸模型。A)極早期HCC(BCLC 0)與對照組比較時microRNA組合的診斷價值的ROC曲線圖。與AFP(AUC=0.68)相比，microRNA組合在區分極早期HCC患者血漿和對照組血漿時具有高得多的診斷準確度(AUC=0.94)。B)早期HCC(BCLC A)與對照組比較時microRNA組合的診斷價值的ROC曲線圖。與AFP(AUC=0.65)相比，microRNA組合在區分早期HCC患者血漿和對照組血漿時具有高得多的診斷準確度(AUC=0.90)。C)中期HCC(BCLC B)與對照組比較時microRNA組合的診斷價值的ROC曲線圖。與AFP(AUC=0.74)相比，microRNA組合在區分中期HCC患者血漿和對照組血漿時具有高得多的診斷準確度(AUC=0.85)。D)進展期HCC(BCLC C)與對照組比較時microRNA組合的診斷價值的ROC曲線圖。與AFP(AUC=0.79)相比，microRNA組合在區分進展期HCC患者血漿和對照血漿時並無顯著差異(AUC=0.80)。 Figure 5 illustrates a preferred microRNA combination comprising at least one target plasma of the invention for identifying different BCLC stages of HCC (hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, Logistic regression models for hsa-miR-26a, hsa-miR-27a and hsa-miR-801). A) ROC plot of the diagnostic value of microRNA combination when very early HCC (BCLC 0) was compared to the control group. Compared to AFP (AUC = 0.68), the microRNA combination had a much higher diagnostic accuracy (AUC = 0.94) in distinguishing between very early HCC patient plasma and control plasma. B) ROC plot of the diagnostic value of microRNA combination when early HCC (BCLC A) was compared to the control group. Compared to AFP (AUC = 0.65), the microRNA combination had a much higher diagnostic accuracy (AUC = 0.90) in distinguishing between early HCC patient plasma and control plasma. C) ROC plot of the diagnostic value of the microRNA combination when the intermediate HCC (BCLC B) was compared to the control group. Compared to AFP (AUC = 0.74), the microRNA combination had a much higher diagnostic accuracy (AUC = 0.85) in distinguishing between mid-stage HCC patient plasma and control plasma. D) ROC plot of the diagnostic value of microRNA combination when advanced HCC (BCLC C) was compared to the control group. Compared to AFP (AUC = 0.79), the microRNA combination did not differ significantly between the plasma of the advanced HCC patients and the control plasma (AUC = 0.80).

圖6說明瞭包含本發明用於鑑定AFP20 ng/ml及AFP>20 ng/ml的HCC的至少一個靶血漿的較佳microRNA組合(hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a和hsa-miR-801)的邏輯回歸模型。A)AFP20 ng/ml的HCC組和對照組比較時microRNA組合的診斷價值的ROC曲線圖。與AFP(AUC=0.63)相比，microRNA組合在區分AFP20 ng/ml的HCC組血漿和對照組血漿時具有高得多的診斷準確度(AUC=0.87)。B)AFP>20 ng/ml的HCC組和對照組比較時microRNA組合的診斷價值的ROC曲線圖。與AFP(AUC=0.69)相比，microRNA組合在區分AFP>20 ng/ml的HCC組血漿和對照組血漿時具有高得多的診斷準確度(AUC=0.90)。 Figure 6 illustrates the inclusion of the present invention for identifying AFP Preferred microRNA combinations of at least one target plasma of 20 ng/ml and AFP > 20 ng/ml of HCC (hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa- Logistic regression models for miR-26a, hsa-miR-27a, and hsa-miR-801). A) AFP ROC plot of the diagnostic value of the microRNA combination when comparing the 20 ng/ml HCC group to the control group. Compared with AFP (AUC=0.63), microRNA combination distinguishes AFP The 20 ng/ml HCC group plasma and control group plasma had much higher diagnostic accuracy (AUC = 0.87). B) ROC plot of diagnostic value of microRNA combination when comparing AFP>20 ng/ml HCC group to control group. Compared to AFP (AUC = 0.69), the microRNA combination had a much higher diagnostic accuracy (AUC = 0.90) in distinguishing between AFP > 20 ng/ml HCC plasma and control plasma.

圖7為7個選出來的microRNA和AFP手術期間變化的箱線圖。分別在術前和術後第6天，取行肝切除手術的54個HCC患者的血液樣本。在術後第6天，AFP和3個microRNA(hsa-miR-21、hsa-miR-192和hsa-miR-223)的表現水準有明顯改變。在外科切除術後，AFP和2個microRNA(hsa-miR-21和hsa-miR-192)的表現水準顯著下降，hsa-miR-223的表現顯著增加。 Figure 7 is a box plot of changes in seven selected microRNAs and AFP procedures. Blood samples from 54 HCC patients undergoing hepatectomy were performed before surgery and on the 6th day after surgery. On the 6th day after surgery, the performance levels of AFP and 3 microRNAs (hsa-miR-21, hsa-miR-192 and hsa-miR-223) were significantly changed. After surgical resection, the performance levels of AFP and two microRNAs (hsa-miR-21 and hsa-miR-192) were significantly decreased, and the performance of hsa-miR-223 was significantly increased.

<110> 復旦大學附屬中山醫院 <110> Zhongshan Hospital affiliated to Fudan University

<120> 由血漿microRNA組合成的肝癌診斷標誌物及一種診斷肝癌的新方法 <120> A diagnostic marker for liver cancer composed of plasma microRNAs and a new method for diagnosing liver cancer

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Claims

一種肝細胞癌診斷標誌物，其係由多種核酸分子組成，每種核酸分子編碼至少一個microRNA序列，所述的多種核酸分子包含編碼hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a、hsa-miR-801以及hsa-miR-1228的八個核酸分子。 A diagnostic marker for hepatocellular carcinoma, which is composed of a plurality of nucleic acid molecules, each nucleic acid molecule encoding at least one microRNA sequence comprising hsa-miR-122, hsa-miR-192, hsa-miR- 21. Eight nucleic acid molecules of hsa-miR-223, hsa-miR-26a, hsa-miR-27a, hsa-miR-801 and hsa-miR-1228.

如申請專利範圍第1項所述的肝細胞癌診斷標誌物，其中所述的hsa-miR-801、hsa-miR-192以及hsa-miR-21的核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調；所述的編碼hsa-miR-122、hsa-miR-26a、hsa-miR-27a以及hsa-miR-223的核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被下調。 The diagnostic marker for hepatocellular carcinoma according to claim 1, wherein the nucleic acid molecules of hsa-miR-801, hsa-miR-192 and hsa-miR-21 are expressed in at least one target plasma. The expression in the at least one control plasma is up-regulated; the nucleic acid molecule encoding hsa-miR-122, hsa-miR-26a, hsa-miR-27a and hsa-miR-223 is in at least one target plasma Performance was down-regulated compared to performance in at least one control plasma.

如申請專利範圍第1項所述的肝細胞癌診斷標誌物，其中所述的編碼hsa-miR-1228的核酸分子在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比不變。 The hepatocellular carcinoma diagnostic marker of claim 1, wherein the nucleic acid molecule encoding hsa-miR-1228 does not exhibit in at least one target plasma as compared to the performance in at least one control plasma. change.

如申請專利範圍第1~3項中任一項所述的肝細胞癌診斷標誌物，其中所述的至少一種對照血漿來自健康個體、慢性B型肝炎患者或肝硬化患者。 The hepatocellular carcinoma diagnostic marker according to any one of claims 1 to 3, wherein the at least one control plasma is from a healthy individual, a chronic hepatitis B patient or a cirrhotic patient.

如申請專利範圍第1~3項中任一項所述的肝細胞癌診斷標誌物，其中所述的肝細胞癌為早期肝細胞癌。 The hepatocellular carcinoma diagnostic marker according to any one of claims 1 to 3, wherein the hepatocellular carcinoma is early hepatocellular carcinoma.

一種肝細胞癌診斷套組，其特徵在於：包含如申請專利範圍第1~3項中任一項所述的肝細胞癌診斷標誌物。 A hepatocellular carcinoma diagnostic kit comprising the hepatocellular carcinoma diagnostic marker according to any one of claims 1 to 3.

如申請專利範圍第6項所述的肝細胞癌診斷套組，其中還包含一個回歸模型，如下：logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-1 92+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209*hsa-miR-801其中，hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a以及hsa-miR-801的表現水準是以hsa-miR-1228為內源性對照檢測得到，模型中的logit(p=HCC)值在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調。 The hepatocellular carcinoma diagnostic kit described in claim 6 of the patent application also includes a regression model as follows: logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR- 1 92+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209*hsa-miR-801 of which hsa-miR-122, hsa The performance levels of -miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR-27a and hsa-miR-801 were measured by endogenous control of hsa-miR-1228 It is obtained that the logit (p=HCC) value in the model is upregulated in at least one target plasma compared to the performance in at least one control plasma.

如申請專利範圍第6項所述的肝細胞癌診斷套組，其中所述的至少一種對照血漿來自健康個體、慢性B型肝炎患者或肝硬化患者。 The hepatocellular carcinoma diagnostic kit of claim 6, wherein the at least one control plasma is from a healthy individual, a chronic hepatitis B patient, or a cirrhotic patient.

如申請專利範圍第6項所述的肝細胞癌診斷套組，其中所述的肝細胞癌為早期肝細胞癌。 The hepatocellular carcinoma diagnostic kit according to the sixth aspect of the invention, wherein the hepatocellular carcinoma is early hepatocellular carcinoma.

一種用於區分至少一個肝細胞癌患者的血漿和至少一個健康個體的血漿的套組，其特徵在於：包含如申請專利範圍第1項所述的肝細胞癌診斷標誌物，其中，所述的至少一種對照血漿來自健康個體。 A kit for distinguishing between plasma of at least one hepatocellular carcinoma patient and plasma of at least one healthy individual, comprising: a hepatocellular carcinoma diagnostic marker according to claim 1 of the invention, wherein At least one control plasma is from a healthy individual.

如申請專利範圍第10項所述的用於區分至少一個肝細胞癌患者的血漿和至少一個健康個體的血漿的套組，其中還包含一個回歸模型，如下：logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209*hsa-miR-801其中，hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a以及hsa-miR-801的表現水準是以hsa-miR-1228為內源性對照檢測得到，模型中的logit(p=HCC)值在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調。 A kit for distinguishing plasma of at least one patient with hepatocellular carcinoma and plasma of at least one healthy individual as described in claim 10, further comprising a regression model as follows: logit (p=HCC)=-1.424 -0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209 *hsa-miR-801, wherein hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR-27a, and hsa-miR-801 The performance level was measured by hsa-miR-1228 as an endogenous control. The logit (p=HCC) value in the model was expressed in at least one target plasma. The performance in one of the less control plasmas was up-regulated.

如申請專利範圍第10項所述的用於區分至少一個肝細胞癌患者的血漿和至少一個健康個體的血漿的套組，其中所述的肝細胞癌為早期肝細胞癌。 The kit for distinguishing between plasma of at least one hepatocellular carcinoma patient and plasma of at least one healthy individual, wherein the hepatocellular carcinoma is early hepatocellular carcinoma, as described in claim 10 of the patent application.

一種用於區分至少一個肝細胞癌患者的血漿和至少一個慢性B型肝炎患者的血漿的套組，其特徵在於：包含如申請專利範圍第1項所述的肝細胞癌診斷標誌物，其中，所述的至少一種對照血漿來自慢性B型肝炎患者。 A kit for distinguishing plasma of at least one patient with hepatocellular carcinoma and at least one patient with chronic hepatitis B, characterized in that it comprises a diagnostic marker for hepatocellular carcinoma as described in claim 1, wherein The at least one control plasma is from a chronic hepatitis B patient.

如申請專利範圍第13項所述的用於區分至少一個肝細胞癌患者的血漿和至少一個慢性B型肝炎患者的血漿的套組，其中還包含一個回歸模型，如下：logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209*hsa-miR-801其中，hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a以及hsa-miR-801的表現水準是以hsa-miR-1228為內源性對照檢測得到，模型中的logit(p=HCC)值在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調。 A kit for distinguishing plasma of at least one patient with hepatocellular carcinoma and plasma of at least one patient with chronic hepatitis B according to claim 13 of the patent application, which further comprises a regression model as follows: logit (p=HCC) =-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR- 27a+0.209*hsa-miR-801 wherein hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR-27a and hsa-miR The performance level of -801 was determined as an endogenous control of hsa-miR-1228, and the logit (p=HCC) value in the model was compared to that in at least one control plasma compared to the performance in at least one control plasma. Up.

如申請專利範圍第13項所述的用於區分至少一個肝細胞癌患者的血漿和至少一個慢性B型肝炎患者的血漿的套組，其中所述的肝細胞癌為早期肝細胞癌。 A kit for distinguishing plasma of at least one patient with hepatocellular carcinoma and plasma of at least one patient with chronic hepatitis B according to claim 13, wherein the hepatocellular carcinoma is early hepatocellular carcinoma.

一種用於區分至少一個肝細胞癌患者的血漿和至少一個肝硬化患者的血漿的套組，其特徵在於：包含如申請專利範圍第1項所述的肝細胞癌診斷標誌物，其中，所述的至少一種對照血漿來自肝硬化患者。 A kit for distinguishing between plasma of at least one patient with hepatocellular carcinoma and plasma of at least one patient with cirrhosis, comprising: a diagnostic marker for hepatocellular carcinoma as described in claim 1 wherein said At least one of the control plasmas is from a patient with cirrhosis.

如申請專利範圍第16項所述的用於區分至少一個肝細胞癌患者的血漿和至少一個肝硬化患者的血漿的套組，其中還包含一個回歸模型，如下：logit(p=HCC)=-1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+0.209*hsa-miR-801其中，hsa-miR-122、hsa-miR-192、hsa-miR-21、hsa-miR-223、hsa-miR-26a、hsa-miR-27a以及hsa-miR-801的表現水準是以hsa-miR-1228為內源性對照檢測得到，模型中的logit(p=HCC)值在至少一種靶血漿中的表現與在至少一種對照血漿中的表現相比被上調。 A kit for distinguishing plasma of at least one patient with hepatocellular carcinoma and plasma of at least one patient with cirrhosis as described in claim 16 of the patent application, which further comprises a regression model as follows: logit (p=HCC)=- 1.424-0.292*hsa-miR-122+0.4511*hsa-miR-192+0.6112*hsa-miR-21-0.1796*hsa-miR-223-0.2487*hsa-miR-26a-0.3542*hsa-miR-27a+ 0.209*hsa-miR-801 wherein hsa-miR-122, hsa-miR-192, hsa-miR-21, hsa-miR-223, hsa-miR-26a, hsa-miR-27a and hsa-miR-801 The performance level was measured with hsa-miR-1228 as an endogenous control, and the logit (p=HCC) value in the model was up-regulated in at least one target plasma compared to the performance in at least one control plasma.

如申請專利範圍第16項所述的用於區分至少一個肝細胞癌患者的血漿和至少一個肝硬化患者的血漿的套組，其中所述的肝細胞癌為早期肝細胞癌。 A kit for distinguishing plasma of at least one patient with hepatocellular carcinoma and plasma of at least one patient with cirrhosis as described in claim 16, wherein the hepatocellular carcinoma is early hepatocellular carcinoma.